sfiA Gene Research Articles

In UV-irradiated Escherichia coli PQ35 overproducing the RecA protein, expression of the sfiA gene and dimer excision are alleviated.

Escherichia coli PQ35 cells carrying the sfiA-::lacZ operon fusion were transformed either with a multicopy plasmid containing the recA gene (pHSG262 recA) or with a multicopy plasmid alone (pHSG262). Both transformants were UV irradiated. Then induction of the sfiA gene and dimer excision were followed. Amplification of the recA gene partly inhibited both sfiA gene induction and dimer excision. The following interpretation of this phenomenon is proposed. When the RecA protein is in abundance, pyrimidine dimers are quickly masked by it. The masked dimers are less efficiently distinguished by excision nuclease and do not provide the induction signal. Due to this, induction of the sfiA gene as well as dimer excision are inhibited early.

Tn10 transposition promotes RecA-dependent induction of a lambda prophage.

We present evidence that Tn10 transposition, or a closely correlated event, induces expression of bacterial SOS functions. We have found that lambda prophage induction is increased in Escherichia coli lambda lysogens containing increased Tn10 transposase function plus single or multiple copies of an appropriate pair of transposon ends. This increase occurs by the normal pathway for prophage induction, which involves RecA-mediated cleavage of the phage lambda repressor protein. We also present evidence that Tn10 promotes induction of expression of the E. coli sfiA gene. Tn10 transposes by a nonreplicative mechanism. We propose that the signal for RecA protease activation and SOS induction is generated by degradation of the transposon donor molecule and suggest that SOS induction is biologically important in helping a cell undergoing transposition to repair and/or recover from damage to the transposon donor chromosome.

Evidence for a specific regulation of recA gene transcription in Escherichia coli

The kinetics of the recA, sfiA and umuDC genes transcription were studied during a double SOS-inducing treatment in Escherichia coli cells using several strains carrying lacZ gene fusions. A transient inhibition in recA, but not in sfiA or umuDC promoted β-galactosidase synthesis was detected after successive UV-irradiations. Results obtained with a recA-lacZ fusion introduced in several DNA-repair mutants demonstrated that neither a lower LexA inactivation nor a decrease in the production of the inducing signal are the events through which the successive UV-irradiation promoted the arrest of recA transcription. On the contrary, a specific UV-dose-dependent delay appears to be the reason for the inhibition of the recA gene transcription in cells irradiated twice.

Inhibition of the SOS response of Escherichia coli by the Ada protein.

Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene. On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant. The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene. Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied. This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity. However, RecF- mutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene.

Induction of the SOS response by hydroxyurea in Escherichia coli K12

Hydroxyurea at concentrations higher than 10 −2 M induced the recA and sfiA genes of E. coli as well as the lambda prophage by a pathway independent of the recBC genes. In addition, the hydroxyurea-mediated induction of the SOS response is accompanied by a recA-dependent decrease on the cellular ATP pool. The presence of the multicopy plasmid pPS2, harboring the nrdAB genes (encoding the ribonucleoside reductase enzyme), abolished the hydroxyurea-induced expression of the recA gene. These data lead us to suggest that induction of the SOS response by hydroxyurea is due to the blocking of DNA replication by the inhibition of the ribonucleoside reductase complex activity.

Modulating action of cyclic AMP on the expression of two SOS genes in Escherichia coli K-12.

Ultraviolet irradiation and cyclic AMP treatment produce a synergistic effect on the induction of the cle1 gene (coding for bacteriocin ColE1) in wild-type strains of Escherichia coli. On the other hand, cyclic AMP does not affect the uv-mediated induction of the recA, sfiA, and umuDC genes. Growth in the presence of glucose or glycerol does not affect the factor of amplification of the expression of the cle1 gene in uv-irradiated cells of the wild-type strain. Although, in cultures not treated with uv, the basal level of cle1 induction is about twice as high in cell grown grown with glycerol as in those using glucose as carbon source. In recA mutants neither simultaneous nor separate treatments with either cyclic AMP or uv irradiation induced transcription of the cle1 gene. Moreover, cyclic AMP induced a slight increase in cle1 gene expression in uv-irradiated cya strains, but not in the crp mutants. Nevertheless, the pattern of the uv-mediated induction of other SOS genes, such as umuDC, was the same in the cya and crp mutants, as in their parental wild-type strains. Furthermore, the uv-mediated induction of lambda prophage was decreased after either addition of cyclic AMP or growth in cultural conditions where the level of this nucleotide was low.

Influence of S9 mix in the induction of SOS system by quercetin

The induction of recA, umuC and sfiA genes by quercetin was studied in the presence and in the absence of S9 mix. The inducing activity of quercetin is higher for sfiA than for recA and umuC genes in the absence of S9 mix. The putative genotoxic metabolites of quercetin produced by S9 mix display different inducing activities of the three SOS genes as compared to quercetin. The induction of sfiA gene is decreased by the presence of S9 mix, whereas an opposite effect was observed concerning umuC and recA. These data suggest that the error-prone repair pathway participates in mutagenesis by quercetin and its metabolites. Moreover, the type of DNA damage exerted by quercetin seems to be determined by its metabolic fate. The importance of testing for the induction of other SOS genes, together with sfiA, in the study of SOS functions as a genotoxic index is emphasized.

Effects of overproduction of single-stranded DNA-binding protein on RecA protein-dependent processes in Escherichia coli

Overproduction of single-stranded DNA-binding protein (SSB) in Escherichia coli led to a decrease in the basal level of repressor LexA. Expression of the LexA-controlled genes was increased differentially, depending on the affinity of the LexA repressor for each promoter: expression of the recA and sfiA genes was increased 5-fold and 1.5-fold, respectively. Despite only a slight effect on expression of sfiA, which codes for an inhibitor of cell division, bacteria overproducing SSB produced elongated cells. In fact, the effect on cell shape appeared to be essentially independent of the expression of the sfiA and recA genes. Bacteria overproducing SSB were therefore phenotypically similar to bacteria partially starved of thymine, in which filamentation results from both sfiA-dependent and sfiA- recA-independent pathways. These data indicate that excess SSB acts primarily by perturbing DNA replication, thereby favoring gratuitous activation of RecA protein to promote cleavage of LexA protein. When bacteria overproducing SSB were exposed to a DNA-damaging agent such as ultraviolet light or mitomycin C, the recA and sfiA genes were fully induced. Induction of the sfiA gene occurred, however, at higher doses in bacteria overproducing SSB protein than in bacteria with normal levels of SSB. Whereas the efficiency of excision repair was apparently increased by excess SSB, the efficiency of postreplication recombinational repair was reduced as judged by a decrease in the recombination proficiency between a prophage and ultraviolet-irradiated heteroimmune infecting phage. Following induction of ssb + bacteria with mitomycin C, the cellular content of SSB was slightly increased. These results provide evidence that SSB modulates RecA protein-dependent activities in vivo. It is proposed that SSB favors the formation of short complexes of RecA protein and single-stranded DNA that mediate cleavage of the LexA and λ repressers, while it delays the formation of long nucleoprotein filaments, thereby slowing down RecA-promoted recombinational events in uninduced as well as in induced bacteria.

The SOS chromotest kit: A rapid method for the detection of genotoxicity

AbstractThe SOS Chromotest is a bacterial assay for genotoxicity, based on the detection of damage to DNA as measured through the SOS DNA repair system. An E. coli strain was derived by fusing the structural lacZ gene with a promoter of one of the SOS genes, namely the sfiA gene. Auxiliary mutations increase the sensitivity of the Chromotest bacteria to genotoxic materials. In the SOS Chromotest, any damage to the DNA results in the activation of the SOS system, including the sfiA gene promoter. This, in turn, induces the de novo transcription and synthesis of the enzyme B. galactosidase. The enzyme is clearly and easily detected by a chromogenic reaction. We have miniaturized the SOS Chromotest and developed it into a self‐contained kit in the microtitration format. The kit contains all the components of the test, including dried bacteria and a dried liver S‐9 mix in a stable state, allowing on‐site operation. The whole test, including the revival of the bacteria, can be completed in 6–7 hours. In addition to being used as a genotoxics detection and monitoring system in chemical, cosmetics, food and pharmaceutical industries, the advantages of the rapid SOS Chromotest can be exploited in full for environmental and occupational health studies. In order to circumvent tedious concentrations of samples (e.g., urine, water), we have tried to further increase the sensitivity of the Chromotest by exposing the bacteria to relatively large volumes of unconcentrated samples. Employing this approach, we were able to demonstrate genotoxic activity in unconcentrated urine samples obtained from people exposed to environmental genotoxins. It appears, therefore, that the SOS Chromotest kit will allow an economically feasible and efficient monitoring of genotoxics in the environment.

Effect of alkylating agents on the expression of inducible genes of Escherichia coli.

Increasing doses of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine, diethyl sulphate and ethylmethane sulphonate cause an inhibition of the expression of the recA and sfiA genes of wild-type Escherichia coli. This behaviour was not observed in a lexA56 mutant which has a defective LexA repressor that is unable to bind to the SOS operator. Furthermore, an ada-1 mutant showed the same behaviour as the wild-type strain indicating that the adaptive proteins are not responsible for the inhibition of recA and sfiA at high doses of alkylating agents. These results suggest that the inhibitory effect of these alkylating agents may be found in the interaction between the LexA repressor and the control regions of sfiA and recA. On the other hand, high doses of either UV light or mitomycin C produced only a slight decrease in the induction of recA and sfiA, whereas bleomycin had no effect. The fact that a repressor structurally related to LexA repressor, such as LacI protein, showed the same behaviour as the LexA repressor when a Lac+ strain was treated with alkylating agents, suggests that these compounds can modify the binding abilities of repressors to DNA, producing a limited or even abolished release of repressors, and so decreasing the expression of inducible genes.

The sfiA11 mutation prevents filamentation in a response to cell wall damage only in a recA+ genetic background.

N-alpha-palmitoyl-L-lysyl-L-lysine dihydrochloride ethyl ester (PLL) at sublethal doses causes filamentous growth of E. coli strains except sfiA mutants, which divide normally in its presence. PLL does not elicit the SOS responses as judged by lambda prophage induction, an increase of RecA protein synthesis or induction of the sfiA operon in a sfiA::lacZ fusion strain. Thus, it appears that filamentation caused by PLL is not an SOS function and might be the result of membrane damage by PLL, which is an amphipathic compound and at higher doses causes cell lysis. This indicates that basal levels of the sfiA gene product are sufficient to inhibit cell division in the presence of PLL. We have found further that the phenotype of the sfiA mutation in the presence of PLL requires a recA+ genetic background and does not occur in E. coli recA1 sfiA11, recA13 sfiA11, recA56 sfiA11 and recA441 sfiA11. All these strains, but rec441 sfiA11, however, regain the ability of sfiA11 mutants to divide in the presence of PLL after transformation with the RecA overproducing-plasmid pXO2. This supports the conclusion that the RecA protein positively affects sfiA11-mediated cell division in the presence of the cell membrane damaging compound, PLL. The basal level of the RecA protein in the recA+ sfiA11 strain is sufficient for this process. An increased level due to overproduction from the multicopy plasmid pXO2 exerts the same effect.

Effect of suppressors of SOS-mediated filamentation on sfiA operon expression in Escherichia coli.

In Escherichia coli, the cell division block observed during the SOS response requires the product of the sfiA gene, whose expression is regulated negatively by the LexA repressor and positively by the RecA protease. We have studied the effect on sfiA expression of sfiA, sfiB, infA, and infB mutations, which are known to affect SOS-associated filamentation. To measure sfiA expression in the different strains, we first constructed a lambda transducing phage carrying an sfiA::lac operon fusion. Mutations at the sfiA locus (dominant and recessive) and the sfiB locus (recessive) had no effect on sfiA expression. The mutations tif (at the recA locus) and tsl (at the lexA locus) are known to induce filamentation and a high level of sfiA expression at 42 degrees C. The infB1 mutation, which suppresses filamentation in a tif tsl strain at 42 degrees C, reduced sfiA expression at 42 degrees C in tif tsl infB1 and tsl infB1 strains but not in a tif infB1 strain. The infA3 mutation, which suppresses tif-mediated filamentation, reduced induction of sfiA expression in a tif infA3 strain at 42 degrees C or after UV irradiation. The isolation and characterization of sfiA constitutive strains revealed only lexA-linked mutations in a sfiA-background, suggesting that LexA is the only readily eliminated repressor of the sfiA gene. Nevertheless, the infA and infB mutations could define elements involved in the regulation of sfiA expression.

How Escherichia coli sets different basal levels in SOS operons

The recA and sfiA genes of Escherichia coli are SOS operons regulated negatively by the LexA repressor. The steady state level of expression of recA is 10-fold higher than that of sfiA, as measured by means of recA::lac and sfiA::lac operon fusions. To study the molecular basis of this difference, we have compared the expression of these two operons in strains in which the concentration of LexA repressor was normal (lexA+), zero (spr amber mutation) or higher than normal (plasmid pJL45, carrying the lexA gene linked to the lac promoter). The results indicate (i) that the recA promoter is about 4 times stronger than the sfiA promoter (as measured in the spr strains), (ii) that neither operon has a physiologically significant level of lexA-independent expression (pJL45 strains), and (iii) that the recA operator has about 2.5 times lower affinity than the sfiA operator for LexA repressor (comparison of lex+ and spr strains). Considering our previous results that the sfiA operon (high operator affinity of LexA) is derepressed very rapidly after inducing treatments and that the recA operon (low operator affinity) is repressed very rapidly when induction is stopped, we conclude that differences in operator affinity do not affect inducibility but serve only to set the basal levels of the different SOS functions.

An inducible DNA replication-cell division coupling mechanism in E. coli.

Cell division is a tightly regulated periodic process. In steady-state cultures of Enterobacteriaceae, division takes place at a well defined cell mass and is strictly coordinated with DNA replication. In wild-type Escherichia coli the formation of cells lacking DNA is very rare, and interruptions of DNA replication arrest cell division. The molecular bases of this replication-division coupling have been elusive but several models have been proposed. It has been suggested, for example, that the termination of a round of DNA replication may trigger a key event required for cell division. A quite different model postulates the existence of a division inhibitor which prevents untimely division and whose synthesis is induced to high levels when DNA replication is perturbed. The work reported here establishes the existence of the latter type of replication-division coupling in E. coli, and shows that the sfiA gene product is an inducible component of this division inhibition mechanism which is synthesized at high levels after perturbations of DNA replication.