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At least 199 records · Page 11Linked to original sources
PubMed2001

Hin recombinase mutants functionally disrupted in interactions with Fis.

A previous genetic screen was designed to separate Hin recombinase mutants into distinct classes based on the stage in the recombination reaction at which they are blocked (O. Nanassy, Zoltan, and K. T. Hughes, Genetics 149:1649-1663, 1998). One class of DNA binding-proficient, recombination-deficient mutants was predicted by genetic classification to be defective in the step prior to invertasome formation. Based on the genetic criteria, mutants from this class were also inferred to be defective in interactions with Fis. In order to understand how the genetic classification relates to individual biochemical steps in the recombination reaction these mutants, R123Q, T124I, and A126T, were purified and characterized for DNA cleavage and recombination activities. Both the T124I and A126T mutants were partially active, whereas the R123Q mutant was inactive. The A126T mutant was not as defective for recombination as the T124I allele and could be partially rescued for recombination both in vivo and in vitro by increasing the concentration of Fis protein. Rescue of the A126T allele required the Fis protein to be DNA binding proficient. A model for a postsynaptic role for Fis in the inversion reaction is presented.

Carrier Proteins↗
PubMed1991

A genetic analysis of Xis and FIS interactions with their binding sites in bacteriophage lambda.

The bacteriophage P22-based challenge-phage system was used to study the binding of Xis and FIS to their sites in attP of bacteriophage lambda. Challenge phages were constructed that contained the X1, X2, and F sites within the P22 Pant promoter, which is required for expression of antirepressor. If Xis and FIS bind to these sites in vivo, they repress transcription from Pant, allowing lysogenization to occur. Challenge phages carrying the XIX2F region in either orientation exhibited lysogenization dependent on both Xis and FIS. Neither Xis nor FIS was capable of functioning by itself as an efficient repressor in this system. This was the first time challenge phages have been constructed that require two different proteins bound simultaneously to act as a repressor. Mutations in the X1, X2, and F sites that inhibit Xis and FIS from binding were isolated by selecting mutant phages that still expressed antirepressor synthesis in the presence of Xis and FIS. DNA sequence analysis of the mutants revealed 38 unique mutations, including single-base-pair substitutions, multiple-base-pair changes, deletions, and insertions throughout the entire X1, X2, and F regions. Some of the mutations verified the importance of certain bases within the proposed consensus sequences for Xis and FIS, while others provided evidence that the DNA sequence outside of the proposed binding sites may affect the binding of the individual proteins or the cooperativity between them.

Attachment Sites, Microbiological↗
PubMed1998

Expression of cyclin D1 in mouse B cell lymphomas of different histologic types and differentiation stages.

The G1 cyclin, cyclin D1, has been implicated in the development of human and mouse tumors. Here we describe immunohistochemical analyses of cyclin D1 for a large panel of mouse B cell tumors. In addition, we characterize cyclin D1 expression in a series of cultured cell lines that represent transformed B cells at different stages of development. Immunohistochemical analysis showed that for low-grade lymphomas, cyclin D1 was expressed by 83% of centroblastic centrocytic (CBCC) and 14% of small lymphocytic lymphomas (SLL). For high-grade tumors, 28% of B lymphoblastic and 23% of centroblastic tumors expressed cyclin D1, while all immunoblastic lymphomas were negative. Studies of RNA and protein prepared from cultured B lineage tumors showed that cyclin D1 was expressed by all pre-B and most B cell tumors but not by cell lines representative of late B cell differentiation or by plasma cells. Expression of cyclin D1 in the lymphomas was not associated with alterations in the genomic structure of the Fis-1 (Bcl-1) common proviral integration site or cyclin D1 itself or with cell growth activity as assessed by expression of proliferating cell nuclear antigen (PCNA).

Animals↗
PubMed1996

Variable structures of Fis-DNA complexes determined by flanking DNA-protein contacts.

The Fis protein from Escherichia coli and Salmonella typhimurium regulates many diverse reactions including recombination, transcription, and replication and is one of the most abundant DNA binding proteins present in the cell under certain physiological conditions. As a specific regulator, Fis binds to discrete sites that are poorly related in primary sequence. Analysis of DNA scission by a collection of Fis conjugates to 1,10-phenanthroline-copper combined with comparative gel electrophoresis has shown that the structures of Fis-DNA complexes are highly variable, displaying overall DNA curvatures that range from < or = 50 degrees to > or = 90 degrees. This variability is primarily determined by differential wrapping of flanking DNA around Fis. By contrast, DNA bending within the core recognition regions appears similar among the binding sites that were analyzed. Flanking DNA contacts by Fis depend on the nucleotide sequence and are mediated by an electrostatic interaction with arginine 71 and a hydrogen bond with asparagine 73, both of which are located outside of the helix-turn-helix DNA binding motif. These contacts strongly influence the kinetics of binding. These data, combined with the crystal structure of Fis, have enabled us to generate new models for Fis-DNA complexes that emphasize the variability in DNA structures within the flanking regions.

Base Sequence↗
PubMed1994

Factor for inversion stimulation-dependent growth rate regulation of individual tRNA species in Escherichia coli.

We have studied the involvement of the factor for inversion stimulation (FIS) in the growth rate-dependent expression of the arginine, leucine, and methionine acceptor tRNA species. The concentration of individual tRNA species relative to 16 S rRNA was determined by blot hybridization using RNA preparations from bacteria with the fis gene deleted and from isogenic wild type bacteria. The RNA preparations were obtained from bacteria growing under steady state conditions in different media. The levels of tRNA(1Leu), tRNA(2Arg), tRNA(4Arg), and tRNA(5Arg decreased in the fis bacteria, relative to the wild type. The difference in levels increased with increasing growth rate. Surprisingly, tRNA(3Leu), tRNA(rMet), and tRNA(eMet) showed the opposite response, with an increase of the tRNA/16 S ratio in the fis bacteria. The tRNA(2Leu, tRNA(4Leu), tRNA(5Leu), and tRNA(3 Arg) had unaffected tRNA/16 S ratios in fis cells. We conclude that FIS, directly or indirectly, is involved in growth rate regulation of some tRNA species and that it affects the composition of the cellular tRNA pool.

Base Sequence↗
PubMed2004

Topological analysis of Hin-catalysed DNA recombination in vivo and in vitro.

In vitro studies have demonstrated that Hin-catalysed site-specific DNA inversion occurs within a tripartite invertasome complex assembled at a branch on a supercoiled DNA molecule. Multiple DNA exchanges within a recombination complex (processive recombination) have been found to occur with particular substrates or reaction conditions. To investigate the mechanistic properties of the Hin recombination reaction in vivo, we have analysed the topology of recombination products generated by Hin catalysis in growing cells. Recombination between wild-type recombination sites in vivo is primarily limited to one exchange. However, processive recombination leading to knotted DNA products is efficient on substrates containing recombination sites with non-identical core nucleotides. Multiple exchanges are limited by a short DNA segment between the Fis-bound enhancer and closest recombination site and by the strength of Fis-Hin interactions, implying that the enhancer normally remains associated with the recombining complex throughout a single exchange reaction, but that release of the enhancer leads to multiple exchanges. This work confirms salient mechanistic aspects of the reaction in vivo and provides strong evidence for the propensity of plectonemically branched DNA in prokaryotic cells. We also demonstrated that a single DNA exchange resulting in inversion in vitro is accompanied by a loss of four negative supercoils.

Bacterial Proteins↗
PubMed2003

Single-cell protein profiling of wastewater enterobacterial communities predicts disinfection efficiency.

The efficiency of enterobacterial disinfection is dependent largely on enterobacterial community physiology. However, the relationship between enterobacterial community physiology and wastewater processing is unclear. The purpose of this study was to investigate this relationship. The influence of wastewater treatment processes on enterobacterial community physiology was examined at the single-cell level by using culture-independent methods. Intracellular concentrations of two conserved proteins, the growth-related protein Fis and the stationary-phase protein Dps, were analyzed by epifluoresence microscopy of uncultivated cells by using enterobacterial group-specific polyclonal fluorochrome-coupled antibodies. Enterobacterial single-cell community protein profiles were distinct for different types of biological treatment. The differences were not apparent when bulk methods of protein analysis were used. Trickling filter wastewater yielded Fis-enriched communities compared to the communities in submerged aeration basin wastewater. Community differences in Fis and Dps contents were used to predict disinfection efficiency. Disinfection of community samples by heat exposure combined with cultivation in selective media confirmed that enterobacterial communities exhibited significant differences in sensitivity to disinfection. These findings provide strategies that can be used to increase treatment plant performance, reduce the enterobacterial content in municipal wastewater, and minimize the release of disinfection by-products into receiving water.

Bacterial Proteins↗
PubMed2005

Fis regulates transcriptional induction of RpoS in Salmonella enterica.

The sigma factor RpoS is known to regulate at least 60 genes in response to environmental sources of stress or during growth to stationary phase (SP). Accumulation of RpoS relies on integration of multiple genetic controls, including regulation at the levels of transcription, translation, protein stability, and protein activity. Growth to SP in rich medium results in a 30-fold induction of RpoS, although the mechanism of this regulation is not understood. We characterized the activity of promoters serving rpoS in Salmonella enterica serovar Typhimurium and report that regulation of transcription during growth into SP depends on Fis, a DNA-binding protein whose abundance is high during exponential growth and very low in SP. A fis mutant of S. enterica serovar Typhimurium showed a ninefold increase in expression from the major rpoS promoter (PrpoS) during exponential growth, whereas expression during SP was unaffected. Increased transcription from PrpoS in the absence of Fis eliminated the transcriptional induction as cells enter SP. The mutant phenotype can be complemented by wild-type fis carried on a single-copy plasmid. Fis regulation of rpoS requires the presence of a Fis site positioned at -50 with respect to PrpoS, and this site is bound by Fis in vitro. A model is presented in which Fis binding to this site allows repression of rpoS specifically during exponential growth, thus mediating transcriptional regulation of rpoS.

Bacterial Proteins↗
PubMed1992

DNA inversions in phages and bacteria.

In certain phages and bacteria, there is a recombination system that specifically promotes the inversion of a DNA fragment. These inversion events appear to act as genetic switches allowing the alternate expression of different sets of genes which in general code for surface proteins. The mechanism of inversion in one class of inversion systems (Gin/Hin) has been studied in detail. It involves the formation of a highly specific nucleoprotein complex in which not only the two recombination sites and the DNA invertase participate but also a recombinational enhancer to which the DNA-bending protein Fis is bound.

Bacteria↗
PubMed1998

Defining the structural and functional roles of the carboxyl region of the bacteriophage lambda excisionase (Xis) protein.

The bacteriophage lambda excisionase (Xis) protein is required for excisive site-specific recombination. Xis is composed of 72 amino acids and binds cooperatively to two DNA sites (X1 and X2) that are arranged as direct repeats. Alternatively, Xis binds cooperatively with the host-encoded factor for inversion stimulation (FIS) protein at the X1 and F sites, respectively. Here we analyzed the effects of missense substitutions from codon 57 to the carboxyl end of the protein and nonsense mutations that truncate the protein at various positions from residues 60 to 69. We find that all of the mutant proteins promote excision to some extent and interact cooperatively with FIS. Some mutants have no detectible phenotype while others are altered in their abilities to promote excision or to interact cooperatively with integrase (Int). Computer modeling predicts that amino acids from residues 59 to 65 are in an alpha-helix conformation. Mutants with substitutions on one side of the helix at residues 57, 60, 63 and 64 as well as truncated mutants containing 60, 61 or 63 amino acids, fail to interact cooperatively with Int suggesting that this region of the protein forms the interface with Int. Mutants with substitutions at other positions in the putative helix have no detectible phenotype. Residues 66 to 68 may form a reverse turn and the last four amino acids (69 to 72) may not be crucial for the structure or function of the protein.

Amino Acid Sequence↗
PubMed1994

The major dimerization determinants of the nitrogen regulatory protein NTRC from enteric bacteria lie in its carboxy-terminal domain.

The NTRC protein (nitrogen regulatory protein C) of enteric bacteria is an enhancer-binding protein that activates transcription by the sigma54-holoenzyme form of RNA polymerase. NTRC is a homodimeric protein that binds to a dyad-symmetrical site in DNA. To activate transcription NTRC must be phosphorylated and must form an appropriate oligomeric species at an enhancer. In order to study subunit exchange between NTRC dimers, we constructed a fusion of the maltose-binding protein (MBP) to the amino-terminal end of NTRC (MBP-NTRC) and visualized the formation of heterodimers between MBP-NTRC and wild-type NTRC by a gel-mobility shift assay for DNA-binding. When MBP-NTRC is mixed with wild-type NTRC at 37 degrees C, subunit exchange occurs rapidly. The apparent half-life for dissociation of homodimers of NTRC is two to three minutes at 37 degrees C and is not changed by phosphorylation. The isolated carboxy-terminal domain of NTRC (91 amino acid residues) forms heterodimers with both wild-type NTRC and MBP-NTRC, indicating that the C-terminal domain is sufficient for dimerization. The apparent rate of dissociation of homodimers of the C-terminal domain is essentially the same as that of full-length NTRC, indicating that the major dimerization determinants of the protein lie in its C-terminal domain. Congruent with this, a truncated form of NTRC from which the last 58 amino acid residues were removed is a monomer in solution. Moreover, truncated forms of NTRC from which the last 16 or 26 amino acid residues were removed are predominantly monomeric in solution, as is a mutant form with the amino acid substitution A410E in its C-terminal domain. Monomerization of the above mutant forms of NTRC can be rationalized on the basis of homology between the C-terminal region of NTRC and a 50 amino acid residue region of the factor for inversion stimulation (FIS) protein.

Bacterial Proteins↗
PubMed1992

The promoter of the tgt/sec operon in Escherichia coli is preceded by an upstream activation sequence that contains a high affinity FIS binding site.

The tgt/sec operon in E. coli consists of five genes: queA, tgt, ORF12, secD, and secF. QueA and Tgt participate in the biosynthesis of the hypermodified t-RNA nucleoside Queuosine, whereas SecD and SecF are involved in protein secretion. Examination of the promoter region of the operon showed structural similarity to promoter regions of the rrn-operons. An upstream activation sequence (UAS) containing a potential binding site for the factor of inversion stimulation (FIS) was found. Gel retardation assays and DNaseI footprinting indicated, that FIS binds specifically and with high affinity to a site centred at position -58. Binding of FIS caused bending of the DNA, as deduced from circular permutation analysis. Various 5' deletion mutants of the promoter region were constructed and fused to a lacZ reporter gene to determine the influence of the UAS element on the promoter strength. An approximately two-fold activation of the promoter by the UAS element was observed.

Base Sequence↗
PubMed2003

Transcription factor as a topological homeostat.

Abundant prokaryotic chromatin architectural proteins often function also as global transcriptional regulators. In addition, some of this class of proteins modulate the activity of cellular topoisomerases and hence, the superhelical density of DNA. The relationships between the global effect of these proteins on DNA topology and their local effects exerted on particular promoter regions remain largely unexplored. One of the best-characterised examples of this class of proteins is the pleiotropic regulator of metabolism FIS, which reduces the activity of DNA gyrase and counteracts the increase of the overall superhelicity of DNA during early exponential growth phase. Binding of FIS to supercoiled DNA molecules in vitro leads to the formation of branched structures and consequent multiplication of apical loops, whereas on bending the upstream regions of stable RNA promoters FIS acts as a topological homeostat maintaining high local levels of supercoiling required for promoter activity. We argue that the coordinated effects of FIS on the global and local DNA architecture optimise gene expression by channelling the free energy of negative supercoiling to specific, biologically relevant sites.

Factor For Inversion Stimulation Protein↗
PubMed1989

Mutational analysis of a prokaryotic recombinational enhancer element with two functions.

The site-specific DNA inversion system Cin encoded by the bacteriophage P1 consists of a recombinase, two inverted crossing-over sites and a recombinational enhancer. The latter approximately 75 bp long genetic element is bifunctional due to its location within the 5' part of the cin gene encoding the recombinase. In order to determine the essential nucleotides for each of its two biological functions we randomly mutated the recombinational enhancer sequence sis(P1) and analysed both functions of the mutants obtained. Three distinct regions of this sequence were found to be important for the enhancer activity. One of them occupies the middle third of the enhancer sequence and it can suffer a number of functionally neutral base substitutions, while others are detrimental. The other two regions occupy the two flanking thirds of the enhancer. They coincide with binding sites of the host-coded protein FIS (Factor for Inversion Stimulation) needed for efficient DNA inversion in vitro. These sequences appear to be highly evolved allowing only a few mutations without affecting either of the biological functions. Taking the effect of mutations within these FIS binding sites into account a consensus sequence for the interaction with FIS was compiled. This FIS consensus implies a palindromic structure for the recombinational enhancer. This is in line with the orientation independence of enhancer action with respect to the crossing-over sites.

Amino Acid Sequence↗
PubMed2006

Fis is required for proper regulation of ssaG expression in Salmonella enterica serovar Typhimurium.

Salmonella pathogenicity island 2 (SPI2) encodes a type III secretion system (TTSS) necessary for bacterial survival and replication in intracellular environment of host cells. SPI2 genes are transcribed preferentially after Salmonella enters the host cells. Transcriptional regulation of ssaG encoding the component of SPI2-TTSS apparatus was studied in vivo and in vitro. Fis, one of the major components of bacterial nucleoid, activated the stationary phase-specific expression of ssaG when Salmonella was grown in LB media. Gel-shift and footprinting analysis showed Fis bound to four distinct sites of the ssaG promoter region with different affinities. All four Fis-binding sites were required for timely transcription activation of ssaG after Salmonella entered macrophage cells. Gentamicin protection experiments using bacteria grown to stationary phase prior to infection showed that the ability of the fis mutant strain to replicate within the RAW264.7 macrophage cells was lower than the wild type. These observations confirm that Fis plays an important role in regulations of SPI2 as well as SPI1 for an efficient regulation of the virulence genes.

Animals↗
PubMed1997

Role of architectural elements in combinatorial regulation of initiation of DNA replication in Escherichia coli.

Bending of DNA is a prerequisite of site-specific recombination and gene expression in many regulatory systems involving the assembly of specific nucleoprotein complexes. We have investigated how the uniquely clustered Dam methylase sites, GATCs, in the origin of Escherichia coli replication (oriC) and their methylation status modulate the geometry of oriC and its interaction with architectural proteins, such as integration host factor (IHF), factor for inversion stimulation (Fis) and DnaA initiator protein. We note that 3 of the 11 GATC sites at oriC are strategically positioned within the IHF protected region. Methylation of the GATCs enhances IHF binding and alters the IHF-induced bend at oriC. GATC motifs also contribute to intrinsic DNA curvature at oriC and the degree of bending is modulated by methylation. The IHF-induced bend at oriC is further modified by Fis protein and IHF affinity for its binding site may be impaired by protein(s) binding to GATCs within the IHF site. Thus, GATC sites at oriC affect the DNA conformation and GATCs, in conjunction with the protein-induced bends, are critical cis-acting elements in specifying proper juxtapositioning of initiation factors in the early steps of DNA replication.

Base Sequence↗
PubMed2004

Expression of cspH upon nutrient up-shift in Salmonella enterica serovar Typhimurium.

The gene cspH, which encodes one of the cold-shock proteins in Salmonella enterica serovar Typhimurium, has previously been reported to be induced during early exponential phase at 37 degrees C. In the present study, the expression of cspH upon nutrient up-shift at 37 degrees C was investigated and found to be affected by DNA gyrase and DNA-binding protein Fis. When cells at stationary phase were subcultured into a rich medium, the mRNA level of cspH increased dramatically prior to the first cell division. However, when the cells were treated with DNA gyrase inhibitors, cspH mRNA was not induced upon nutrient up-shift. The low level of DNA superhelical density at the cspH promoter in part affected the expression of cspH mRNA in vitro. In addition, a fis-deficient strain had a lower level of cspH mRNA than the wild-type upon nutrient up-shift. Finally, a cspH-lacZ construct, in which the putative binding region for Fis was deleted in the cspH promoter, expressed a low level of LacZ, in contrast to the native cspH-lacZ construct.

Bacterial Proteins↗
PubMed2003

Molecular flip-flops formed by overlapping Fis sites.

The DNA-binding protein Fis frequently uses pairs of sites 7 or 11 base pairs (bp) apart. Two overlapping Fis sites separated by 11 bp are found in the Escherichia coli origin of chromosomal replication. Only one of these sites is bound by Fis at a time, so the structure is a molecular flip-flop that could direct alternative firing of replication complexes in opposite directions. Alternatively, the flip-flop could represent part of an on-off switch for replication. Because they can be used to create precise switched states, molecular flip-flops could be used as the basis of a novel molecular computer.

Bacterial Proteins↗