sulA Gene Research Articles

Antimicrobial and Antioxidant Activity of Some Nitrogen-Containing Heterocycles and Their Acyclic Analogues.

We investigated antimicrobial and antioxidant activity of nitrogen-containing heterocycles and their acyclic analogues, some of which can be considered as promising in terms of biological activity. Based on structure, 26 tested compounds were divided into 4 groups. In the test with 2,2-diphenyl-1-picrylhydrazyl (DPPH), the compounds of the group 2 had the highest radical-binding activity (RBA) (53-78%), while those of group 3 had the lowest values (1.5-5.2%). In oxygen radical absorbance capacity assay, all compounds from groups 1, 2 and 3 showed high RBA: 44-94% at 50µM. The highest bacteriostatic activity against Escherichia coli was found for four compounds in group 2 (MIC = 0.25-1mM) and low bacteriostatic activity for group 3 (MIC > 4mM). Some relationships between the structure of compounds and the values of the MIC are revealed. It was also found that four substances from different groups had the ability to inhibit the formation of colonies in E. coli from 1.3 to 5.7 times. Four compounds reduced specific biofilm formation by 40-60%. The tested substances did not induce the expression of the sulA gene controlled by the SOS system, which indicates the lack of genotoxic activity. None of the tested compounds had pro-oxidant activity. This was shown by both the absence of production hydrogen peroxide in a bacteria-free medium and inability to induce expression of the katG gene encoding HPI catalase in growing E. coli. The data obtained could be useful in the development of new drugs.

Preliminary study on phenotype and genotype of antibiotic resistance in Salmonella enterica subsp. enterica serotype Typhimurium Isolated from Chicken Samples in Malaysia

Salmonellosis is a significant public health concern around the world. The issue of antibiotic resistance, particularly in non-typhoidal Salmonella (NTS), is a global threat to human and animal health. Thirty-five Salmonella enterica subsp. enterica serotype Typhimurium (S. Typhimurium) isolated from various chicken samples were tested for antimicrobial-resistant profiling using the disc diffusion method. The strains were further examined for the presence of the resistance genes by using polymerase chain reaction (PCR) according to published protocols. Phenotype resistance profiling revealed that 22.9% (8 out of 35) isolates showed resistance to five antibiotics used in this study. All the penta-resistant isolates carried the blaTEM, floR, strA, and tetA gene which encoded for ampicillin, chloramphenicol, streptomycin, and tetracycline resistance. Only one isolate (1 out of 35) was found to contain the sulfonamides resistance, sulA gene. Nine isolates were found susceptible to all antibiotics tested and do not harbor any of the resistant associated genes. These findings provide evidence that the presence of resistance genes contributes to the phenotypic resistance profile, and thus could give rise to the emergence of the drug-resistant strain of Salmonella.

Regulation of ssb Gene Expression in Escherichia coli

Bacterial SSB proteins, as well as their eukaryotic RPA analogues, are essential and ubiquitous. They avidly bind single-stranded DNA and regulate/coordinate its metabolism, hence enabling essential DNA processes such as replication, transcription, and repair. The prototypic Escherichia coli SSB protein is encoded by an ssb gene. Although the ssb gene promoters harbor an SOS box, multiple studies over several decades failed to elucidate whether ssb gene expression is inducible and SOS dependent. The SOS regulon is comprised of about 50 genes, whose transcription is coordinately induced under stress conditions. Using quantitative real-time PCR, we determined the ssb gene expression kinetics in UV- and γ-irradiated E. coli and revealed that ssb gene expression is elevated in irradiated cells in an SOS-dependent manner. Additionally, the expression of the sulA gene was determined to indicate the extent of SOS induction. In a mutant with a constitutively induced SOS regulon, the ssb gene was overexpressed in the absence of DNA damage. Furthermore, we measured ssb gene expression by droplet digital PCR during unaffected bacterial growth and revealed that ssb gene expression was equal in wild-type and SOS− bacteria, whereas sulA expression was higher in the former. This study thus reveals a complex pattern of ssb gene expression, which under stress conditions depends on the SOS regulon, whereas during normal bacterial growth it is unlinked to SOS induction. The E. coli ssb gene is SOS regulated in such a way that its basal expression is relatively high and can be increased only through stronger SOS induction. The remarkable SOS induction observed in undisturbed wild-type cells may challenge our notion of the physiological role of the SOS response in bacteria.

Class I Polyhydroxyalkanoate (PHA) Synthase Increased Polylactic Acid Production in Engineered Escherichia Coli.

Polylactic acid (PLA), a homopolymer of lactic acid (LA), is a bio-derived, biocompatible, and biodegradable polyester. The evolved class II PHA synthase (PhaC1 Ps6-19) was commonly utilized in the de novo biosynthesis of PLA from biomass. This study tested alternative class I PHA synthase (PhaC Cs ) from Chromobacterium sp. USM2 in engineered Escherichia coli for the de novo biosynthesis of PLA from glucose. The results indicated that PhaC Cs had better performance in PLA production than that of class II synthase PhaC1 Ps6-19. In addition, the sulA gene was engineered in PLA-producing strains for morphological engineering. The morphologically engineered strains present increased PLA production. This study also tested fused propionyl-CoA transferase and lactate dehydrogenase A (fused Pct Cp /LdhA) in engineered E. coli and found that fused Pct Cp /LdhA did not apparently improve the PLA production. After systematic engineering, the highest PLA production was achieved by E. coli MS6 (with PhaC Cs and sulA), which could produce up to 955.0 mg/L of PLA in fed-batch fermentation with the cell dry weights of 2.23%, and the average molecular weight of produced PLA could reach 21,000 Da.

Real-time мониторинг физиологических параметров для изучения раннего ответа бактерий Escherichia coli на пероксидный стресс

The response of aerobically growing Escherichia coli cultures to peroxide stress was studied using synchronous real-time monitoring of parameters pO2 (oxygen partial pressure), pH, Eh (redox potential of the culture), extracellular levels of K+ and S2-, in combination with traditional physiological, biochemical and genetic methods. A sharp increase in pO2 level in the medium caused by the destruction of H2O2 by endogenous catalases was observed in the early response to peroxide stress. The addition of 100 µM H2O2 provoked a reversible decrease in the specific growth rate, accompanied by an increase of extra- and intracellular glutathione and a short-term increase in sulfide production. Treatment with a high dose of H2O2 (10 mM) led to growth inhibition and a CFU loss, most pronounced in glutathione synthesis mutants. Simultaneously, in 8% of cells, the membrane potential decreased, a part of potassium was released into the medium, and the expression of the sulA gene, which is part of the SOS-regulon, increased. Application of an integrated approach to the study of stress revealed a close relationship between growth parameters (specific growth rate and survival), respiratory activity, sulfide production, and the ability of E. coli cells to maintain the membrane potential and K+ gradient.

Cell Lysis Directed by SulA in Response to DNA Damage in Escherichia coli.

The SOS response is induced upon DNA damage and the inhibition of Z ring formation by the product of the sulA gene, which is one of the LexA-regulated genes, allows time for repair of damaged DNA. On the other hand, severely DNA-damaged cells are eliminated from cell populations. Overexpression of sulA leads to cell lysis, suggesting SulA eliminates cells with unrepaired damaged DNA. Transcriptome analysis revealed that overexpression of sulA leads to up-regulation of numerous genes, including soxS. Deletion of soxS markedly reduced the extent of cell lysis by sulA overexpression and soxS overexpression alone led to cell lysis. Further experiments on the SoxS regulon suggested that LpxC is a main player downstream from SoxS. These findings suggested the SulA-dependent cell lysis (SDCL) cascade as follows: SulA→SoxS→LpxC. Other tests showed that the SDCL cascade pathway does not overlap with the apoptosis-like and mazEF cell death pathways.

A marked Copy of the Manuscriptinfluence of Oxyr on Susceptibility of Planktonic and Sessile Escherichia Coli Cultures to Ciprofloxacin and Cefotaxime

Alternation of bacterial antioxidant defense pathways might affect susceptibility to antibiotics in dual ways. Using a relatively simple model based on wild-type and oxyR Escherichia coli mature biofilms, their counterpart planktonic cultures and exponentially growing planktonic cultures, we explored the role of OxyR-mediated metabolism alternations in modulation of susceptibility to antibiotics ciprofloxacin and cefotaxime. All three types of cultures were placed in fresh medium,1 h after antibiotics were added and incubation continued further for 2 h. Killing rates of antibiotics were determined, biofilm eradication using crystal violet assay was estimated, expression of rpoS, katG, sulA genes as well as HPI and HPII catalase activity were measured. Biofilms of both strains were more recalcitrant to ciprofloxacin and cefotaxime at all tested concentrations compared to exponentially growing planktonic cultures. In oxyR biofilms killing rate of ciprofloxacin was lower, and killing rate of cefotaxime was higher compared to the parental strain. Compared to biofilms, wild-type biofilm counterpart planktonic cultures showed higher tolerance to low doses of ciprofloxacin, while oxyR plankton demonstrated higher tolerance to cefotaxime. Higher recalcitrance of oxyR biofilms to ciprofloxacin may be caused by an increase in persister cells under conditions of enhanced oxidative stress and activated SOS response.

Gamma irradiation triggers a global stress response in Escherichia coli O157:H7 including base and nucleotides excision repair pathways

Shiga toxin-producing Escherichia coli O157:H7, one of the most severe human foodborne pathogens, can withstand several stresses, including some levels of γ-irradiation. In this study, the response of E. coli O157:H7 to a sensitization irradiation dose of 0.4 kGy was assessed using RNA-seq transcriptomic at 10 (t10) and 60 (t60) min post-irradiation, combined with an isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis at 60 min post-irradiation. Several functions were induced by the treatment, such as base excision repair and nucleotide excision repair pathways; sulfur and histidine metabolism, and virulence mechanisms. Additionally, the sulA gene, coding for the cell division repressor, together with other genes involved in SOS response and repair mechanism (including recA, recN, recJ, recQ, mutM and uvrB) were up-regulated at t60. As the early response to irradiation stress (t10), dnaK, groEL, ibpA, sulfur metabolism genes, as well as those related to oxidative stress were up-regulated, while histidine biosynthesis genes were down-regulated. Acid stress, heat shock, UV resistance and several virulence genes, especially stx2A/stx2b which code for the Shiga toxins characteristic of O157:H7, were upregulated at 60 min post-irradiation. The treatment was also found to increase the levels of CysN, MutM, DinG and DnaC in the cells, proteins involved respectively in sulfur metabolism, base excision repair, recombinational DNA repair and chromosome replication. Our results provide insights into the resistance response of E. coli O157:H7 to a non-lethal irradiation dose. Our findings indicate that E. coli O157:H7 can resist to γ-irradiation through important modifications in genes expression and proteins profiles.

Effects of voltage on sulfadiazine degradation and the response of sul genes and microbial communities in biofilm-electrode reactors

Few studies have been performed on both the potential and the risks of biofilm-electrode reactors (BERs) with regard to the removal of antibiotics. This study used 33 BERs to investigate the removal rate and degradation pathway of sulfadiazine (SDZ). Furthermore, the effects of additional electrons on sul genes and microbial community composition were examined. The study found that rapid elimination rates of 20mg/L SDZ were observed during the first 3h with different DC voltage rates. Even high concentrations (160mg/L) could be rapidly removed after 24h of system operation. Pyrimidin-2ylsulfamic acid and aniline were noted to be principal products, and an SDZ degradation mechanism was proposed. The study identified 41 species of microorganism; based on bacterial community divergence caused by voltage, and six samples were grouped into four clusters. The relative abundances of sul genes from biofilm were in the following order: sulII >sulIII >sulI >sulA. The sulI, sulII, and sulA genes were enhanced with electrical stimulation in the cathode layer. It is noteworthy that sul genes were not detected in the effluent after 24h of operation.

The effect of 20-hydroxyecdysone on the susceptibility of Escherichia coli to different antibiotics.

The aim of this study was to investigate the influence of 20-hydroxyecdysone (20E) on the susceptibility of growing Escherichia coli to antibiotics. Susceptibility of E.coli to antibiotics in the presence of 20E was estimated by determination of the colony-forming ability and the specific growth rate. Pretreatment with 20E decreased the bactericidal effect of ciprofloxacin (0·3 and 3·0μgml-1 ), streptomycin (10 and 40μgml-1 ) and kanamycin (10μgml-1 ) and increased the bactericidal action of 0·03μgml-1 ciprofloxacin. To study the influence of 20E on gene expression, we used strains of E.coli carrying fusions of promoters of relevant genes with the structural gene of β-galactosidase. 20E had no marked effect on the expression of antioxidant genes katG, katE, sodA and the rpoS gene (general stress response), while it (alone or combined with ciprofloxacin) markedly stimulated expression of the sulA gene belonging to the SOS regulon (DNA damage response). In growing E.coli, 20E modulated the bactericidal action of antibiotics and stimulated the SOS response. The results of this study may be used to enhance the efficiency of antibacterial therapies.

Engineering the growth pattern and cell morphology for enhanced PHB production by Escherichia coli.

E. coli JM109∆envC∆nlpD deleted with genes envC and nlpD responsible for degrading peptidoglycan (PG) led to long filamentous cell shapes. When cell fission ring location genes minC and minD of Escherichia coli were deleted, E. coli JM109∆minCD changed the cell growth pattern from binary division to multiple fissions. Bacterial morphology can be further engineered by overexpressing sulA gene resulting in inhibition on FtsZ, thus generating very long cellular filaments. By overexpressing sulA in E. coli JM109∆envC∆nlpD and E. coli JM109∆minCD harboring poly(3-hydroxybutyrate) (PHB) synthesis operon phbCAB encoded in plasmid pBHR68, respectively, both engineered cells became long filaments and accumulated more PHB compared with the wild-type. Under same shake flask growth conditions, E. coli JM109∆minCD (pBHR68) overexpressing sulA grown in multiple fission pattern accumulated approximately 70% PHB in 9g/L cell dry mass (CDM), which was significantly higher than E. coli JM109∆envC∆nlpD and the wild type, that produced 7.6 g/L and 8g/L CDM containing 64 % and 51% PHB, respectively. Results demonstrated that a combination of the new division pattern with elongated shape of E. coli improved PHB production. This provided a new vision on the enhanced production of inclusion bodies.

Survival and SOS response induction in ultraviolet B irradiated Escherichia coli cells with defective repair mechanisms

Purpose In this paper, the contribution of different genes involved in DNA repair for both survival and SOS induction in Escherichia coli mutants exposed to ultraviolet B radiation (UVB, [wavelength range 280–315 nm]) was evaluated.Materials and methods E. coli strains defective in uvrA, oxyR, recO, recN, recJ, exoX, recB, recD or xonA genes were used to determine cell survival. All strains also had the genetic sulA::lacZ fusion, which allowed for the quantification of SOS induction through the SOS Chromotest.Results Five gene products were particularly important for survival, as follows: UvrA > RecB > RecO > RecJ > XonA. Strains defective in uvrA and recJ genes showed elevated SOS induction compared with the wild type, which remained stable for up to 240 min after UVB-irradiation. In addition, E. coli strains carrying the recO or recN mutation showed no SOS induction.Conclusions The nucleotide excision and DNA recombination pathways were equally used to repair UVB-induced DNA damage in E. coli cells. The sulA gene was not turned off in strains defective in UvrA and RecJ. RecO protein was essential for processing DNA damage prior to SOS induction. In this study, the roles of DNA repair proteins and their contributions to the mechanisms that induce SOS genes in E. coli are proposed.

Engineering Escherichia coli for enhanced production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in larger cellular space

Polyhydroxyalkanoates (PHA) are intracellularly accumulated as inclusion bodies. Due to the limitation of the cell size, PHA accumulation is also limited. To solve this problem, Escherichia coli was enlarged by over-expression of sulA gene to inhibit the cell division FtsZ ring assembly, leading to the formation of filamentary E. coli that have larger internal space for PHA accumulation compared with rod shape E. coli. As a result, more than 100% increases on poly(3-hydroxybutyrate) (PHB) contents and cell dry weights (CDW) were achieved compared with its control strain under same conditions. The enlarged cell strategy was applied to the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) or P(3HB-co-4HB) by sad, gabD, essential genes ispH and folK knockout E. coli harboring two addictives and thus stable plasmids consisting of P(3HB-co-4HB) producing genes, including phaCAB operon, orfZ, 4hbD, sucD, essential genes ispH and folK as well as the sulA. The so constructed E. coli grew in glucose to form filamentary shapes with an improved P(3HB-co-4HB) accumulation around 10% more than its control strain without addition of 4HB precursor, reaching over 78% P(3HB-co-4HB) in CDW. Importantly, the shape changing E. coli was able to precipitate after 20min stillstand. Finally, the filamentary recombinant E. coli was not only able to produce more P(3HB-co-4HB) from glucose but also allow convenient downstream separation from the fermentation broth.

The role of thiol redox systems in the resistance of Escherichia coli in the stationary phase

The effect of glutathione (gshA), thioredoxin (trxA), and thioredoxin reductase (trxB) mutations on the adaptation of Escherichia coli to prolonged glucose starvation was investigated. The trxB mutation had the worst consequences for the stationary-phase cells. These bacteria exhibited decreased survival, increased sensitivity to oxidants, and decreased expression of the katE and sulA genes. As the stationary phase proceeded, the physiological resistance to antibiotics increased in all the strains tested; however, the thiol redox system mutants were far less able to develop antibiotic resistance than the parent strain cells. During the stationary phase, a considerable shift was observed in the redox status of intra- and extracellular glutathione toward the oxidative values. These results indicate that the thiol redox systems play an important role in the adaptation of Escherichia coli to prolonged starvation and antibiotic resistance.

The role of thiol redox systems in the response of Escherichia coli to far-UV irradiation

Escherichia coli mutants deficient in glutathione (gshA), glutaredoxin (grxA), thioredoxin (trxA), and thioredoxin reductase (trxB) synthesis were studied with respect to their resistance to far-UV (UV254) exposure. The trxA, trxB, and grxA mutants subjected to a short-term UV exposure were found to be more resistant to UV irradiation than the parent cells. Under the same conditions, the trxA and trxB mutants demonstrated a high level of induction of the sulA gene, a component of the SOS regulon. The mutagenic effect of long-term UV exposure of all the mutants with redox deficiencies was more pronounced than in the case of the parent strain, and the trxA and trxB mutants were found to be the least viable microorganisms. Pretreatment of the cells with low concentrations of the thiol-oxidizing agent diamide enhanced the sulA gene expression; however, high concentrations of diamide inhibited sulA expression. The data obtained indicate that the thiol redox systems of E. coli are involved in its response to far-UV irradiation.

The role of thiol redox systems in the peroxide stress response of Escherichia coli

The effect of mutations in the genes encoding glutathione, glutaredoxin, thioredoxin, and thioredoxin reductase on the response of growing Escherichia coli to oxidative stress was studied. The gshA mutants defective in glutathione synthesis had the lowest resistance to high doses of H2O2, whereas the trxB mutants defective in thioredoxin reductase synthesis had the highest resistance to this oxidant, exceeding that of the parent strain. Among the studied mutants, the trxB cells demonstrated the highest basic levels of catalase activity and intracellular glutathione; they were able to rapidly reach the normal GSH level after oxidative stress. At the same time, these bacteria showed high frequency of induced mutations. The expression of the katG and sulA genes suggests that, having different sensitivity to high oxidant concentrations, the studied mutants differ primarily in their ability to induce the antioxidant genes of the OxyR and SOS regulons.

Radioprotective effect of sodium diethyldithiocarbamate (DDC) and S-2-aminoethyl-isothioronicadenosin-5-triphosphate (adeturon) in γ-irradiated Escherichia coli cells

The effect of sodium diethyldithiocarbamate (DDC) and S-2-aminoethyl-isothiouronicadenosin-5-triphosphate (adeturon) in the induction of Escherichia coli SOS response promoted by γ-irradiation was studied by measuring the induction of sulA gene and the induction of lambda prophage. Furthermore, as a way of measure the exonuclease activity in γ-irradiated cells in the presence or absence of both compounds, the DNA degradation was determined. Adeturon did not affected DNA degradation, but inhibited the induction of the SOS functions studied. On the contrary, DDC inhibited DNA degradation as well as the induction of the sulA gene, but enhanced lambda induction in E. coli lysogenic strains. These results indicate that both compounds diminish the DNA damage produced by γ-irradiation and also suggest that the mechanisms of radioprotection must be different. Thus, radioprotection mediated by DDC should involve free hydroxyl radical scavenging and a minor activity of exonuclease. The enhancement of phage induction in E. coli cells that DDC produces could be attributed to its quelant effect and this would not be not probably directly related to radioprotection. Adeturon, as thiols, may serve also as scavenging agent of free hydroxyl radicals, diminishing indirectly the DNA damage level. In addition, adeturon must interact with DNA in the same form that other aminothiol compounds do it. This interaction, mediated by amino groups of adeturon, may serve to concentrate these compounds near of the DNA damage site, increasing the potential for the thiol portion of the molecule to donate hydrogen, decreasing the damage level on DNA molecule. However, adeturon do not modify the exonuclease activity. Some topic about the possible clinical application of both compounds are discussed.

P53-mediated protective responses to UV irradiation.

Exposure of cells to DNA-damaging agents elicits a complex set of acute cellular responses. Generally, DNA damage-inducible (DDI) responses serve to protect the cell from genotoxic adversity. In Escherichia coli, the SOS regulon frequently has been studied by using UV radiation as a model-inducing agent (1). In this prototypical response, approximately 20 genes are coordinately induced that have roles in DNA repair, mutagenesis, recombination, and growth control (1). This general theme, involving the coupling of cell cycle arrest and DNA repair by coordinate induction of several genes, appears to be conserved between single-cell organisms and mammalian cells; for example, the sulA gene, a component of the SOS response in E. coli, encodes a growth inhibitor analogous to cell cycle inhibitors of mammalian cells (reviewed in ref. 2). A recent example of interspecies conservation of cell cycle checkpoint functions was shown in the case of Schizosaccharomyces pombe Chk1 protein kinase and its human homolog, both of which elicit a G2/M checkpoint response (3). In yeast, 80 or more genes are DDI, representing almost 1% of the genes in this organism (4).

The Salmonella sulA-test: a new in vitro system to detect genotoxins

The Salmonella sulA-test is a newly developed colorimetric assay to detect genotoxins. This technique is based on the ability of DNA-damaging agents to induce the sulA gene, one of the SOS response genes. A constructed plasmid, pEM1968, carrying a fused sulA′::′lacZ was introduced into Salmonella typhimurium TA1538. Monitoring sulA gene expression was performed by assaying the ß-galactosidase activity in the transformed strain S. typhimurium TA1538/pEM1968. A simple, fast and sensitive liquid incubation procedure has been developed after optimization of the S9 mix composition and ß-galactosidase assay. The SOS-inducing potency (SOSIP, μM −1) was defined as the slopes of the non-linear dose-response relationships. Twenty-one chemicals with different modes of action were examined for a preliminary evaluation of the test. Nineteen chemicals were genotoxic in the Salmonella sulA-test. The SOSIP ranged from 1.2 · 10 −4 μM −1 (ethyl methanesulfonate) to 419.9 μM −1 (bleomycin). Sodium azide and 5-fluoroucil were not genotoxic. Frameshift, base-pair and oxidative genotoxins were detected by the tester strain. The calculated SOSIP and the minimum concentrations detected (MCD) in the Salmonella sulA-test were compared to the reported values obtained with two similar assays: the SOS Chromotest and umu-test. The SOSIP values of 12 compounds were the highest in this new assay. Five chemicals tested in the Salmonella sulA-test gave similar SOSIP values with those of one of the two other tests. ICR-191 had the highest SOSIP with the SOS Chromotest and 3-methylchloranthrene showed the highest SOSIP with the umu-test. Similarly, the lowest MCD values were found for 12 compounds in the Salmonella sulA-test. Four compounds had close MCD values in this assay and one of the two other techniques. The SOS Chromotest remained the most sensitive assay for cisplatin and ICR 191. The umu-test was the technique of choice for 3-methylchloranthrene.

Overproduction and purification of SulA fusion protein in Escherichia coli and its degradation by Lon protease in vitro.

To overproduce extremely unstable SulA protein, which is the cell-division inhibitor of Escherichia coli, we fused the sulA gene to the maltose-binding protein (MBP) fusion vectors with or without the signal sequence (plasmids pMAL-p-SulA and pMAL-c-SulA respectively). The amount of the full-length fusion protein expressed from the plasmid pMAL-p-SulA (pre-MBP-SulA) in E. coli was much larger than that expressed from the plasmid pMAL-c-SulA (MBP-SulA). A major amount of the pre-MBP-SulA fusion protein was expressed in a soluble form and affinity-purified by amylose resin. Since site-specific cleavage of the fusion protein with factor Xa resulted in the precipitation of SulA protein, the pre-MBP-SulA fusion protein was used to study the degradation of SulA protein by E. coli Lon protease in vitro. It was found that only the SulA portion of the fusion protein was degraded by Lon protease in an ATP-dependent manner. This result provides direct evidence that Lon protease plays an important role in the rapid degradation of SulA protein in cells.