The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing 8-azaguanine

Low concentrations (?20 μg/ml) of 8-azaguanine are 1000 fold more toxic to V79 Chinese hamster cells in medium containing 10% dialyzed fetal calf serum than in medium containing 10% undialyzed serum. Serum enzyme activity that converts AG to nontoxic 8-azaxanthine degrades AG at the same rate, whether or not the serum is dialyzed. However, cytotoxicity results similar to those obtained with US were produced in medium containing DS and 2.5 μg of hypoxanthine (HX)/ml (DSH). Therefore, serum HX is considered to be responsible for the relatively low cytotoxicity of AG in medium containing US. Colonies that arose in medium containing AG were isolated and characterized. Those that remained resistant to AG (40 μg/ml) and sensitive to aminopterin in the presence of HX and thymidine (HAT) were considered mutants; nonmutants were sensitive to AG and resistant to HAT. Colonies isolated from medium containing DSH of US and low concentrations of AG were not mutants, but those from medium containing high concentrations (? μg/l) of AG were mutants. Spontaneous and N-methyl-N′-nitrosoguanidine induced mutants were detectable in medium containing DSH without replating the cells prior to adding AG (?30 μg/ml), but in order to detect MNNG induced mutations in medium containing DS replating was essential. In DS, the mutation frequency increased as an exponential function of the toxicity of MNNG, but remained two orders of magnitude lower than the induced mutation frequencies that occurred in DSH, HX, in DSH or US, produced profound effects, other than interference with AG toxicity, that distort the results of mutagenesis assays. To study mutation using AG resistance as the endpoint, it is essential to use dialyzed serum.     

Further evidence for the genetic origin of mutations in mammalian somatic cells: the effects of ploidy level and selection stringency on dose-dependent chemical mutagenesis to purine analogue resistance in Chinese hamster ovary cells.

Whether resistance to purine analogues 8-azaguanine (AG) and 6-thioguanine (TG) in mammalian cells is due to gene mutation or to epigenetic changes was investigated by an ethyl methanesulfonate (EMS) dose-dependent induced “resistance” to these analogues in two near-diploid (2N) and one tetraploid (4N) Chinese hamster ovary (CHO) cells. EMS produced higher cell killing in 2N than in 4N cells. In the 2N cells, EMS-induced mutations to TG (1.7 μg/ml) resistance increased approximately as a linear function of the dose from 0–400 μg/ml. However, EMS was ineffective in inducing such mutation in the 4N cells. These observations are consistent with the notion that the induced TG resistance arose as a result of mutation at the gene or chromosome level. In each cell type, both the “observed” spontaneous and the EMS-induced frequency to purine analogue resistance decreased with increasing concentration of purine analogues. However, among the “resistant” clones a high proportion of those selected at 1.2 and 3.0 μg/ml of AG, a small portion selected at 7.5 μg/ml of AG, and virtually none at 1.7 and 6.0 μg/ml of TG are capable of growth in medium containing aminopterin (10 μM). This suggests that, under less stringent selective conditions, some resistant variants were being selected through mechanisms not yet defined.     

Genetic Transformation of Leaf Explants of Populus tomentosa

The leaf disc method developed by Horsch et al. (1985) has been used for transformation of Populus tomentosa. The strain of Agrobacterium tumefaciens used harbored a reconstructed Ti plasmid which contained gene 4 of T–DNA and the chimeric CAT(chloramphenicol acetyltransferase) gene. Leaf explants from shoot cultures of Populus tomentosa were co-culfivated with the bacterium. On the hormone free medium, teratoma-like shoots developed from the edge of the leaf explants. When the abnormal shoots were excised from the explants and transferred onto rooting medium, a mass of callus formed at the base of shoots, with new shoots developing, but without root formation. The measurement of'endogenous cytokinin showed that the transformed shoots produced 14 times as much iso-pentenyl adenosine as untransformed shoots did. All teratoma-like shoots-tested showed the presence of nopaline, and were able to grow well. on the medium containing 60-100μg/ml chloromycetin, while normal shoots turned white after 40 days. Pretreatment of A. tumefaciens with phenolic compound, salicylic acid, would increase the frequency of transformation significantly.     

Resistance system of Mycobacterium bovis B.C.B. to aminoglycoside- and peptide-antibiotics. Comparison of resistant phenotypes between Mycobacterium tuberculosis and Mycobacterium bovis

The resistance system of Mycobacterium bovis (B.C.G.) to aminoglycoside-and peptide-antibiotics has been studied. The phenotype of mutants isolated from the parent B.C.G. strain by a single-step selection with an antibiotic were classified into the following three types: (1) resistant only to a low concentration (200 μg/ml) of kanamycin in Ogawa egg medium (k1R); (2) resistant to a low concentration (200 μg/ml) of viomycin and of capreomycin (2R); and (3) resistant to a high concentration (1,000 μg/ml or more) of kanamycin and low concentrations (100 to 200 μg/ml) of lividomycin and of paromomycin (KR). The mutants showing these phenotypes, k1R, 2R, and KR, were isolated from the parent strain by inoculating the strain into media containing 100 μg/ml of kanamycin, and 100 μ/g/ml of viomycin or capreomycin, and 1,000 μg/ml of kanamycin, respectively, at rates of 10^?5-10^?6, 10^?5-10^?6, and 10^?6-10^?7, respectively, in a total viable population of the parent strain. Unlike in the case of M. tuberculosis, no mutant could be isolated from the parent strain by use of enviomycin, lividomycin, and/or paromomycin. In contrast to the fact that quadruply resistant mutants were isolated directly from the parent H37Rv strain of M. tuberculosis, such mutants could be isolated only by two-step selections. Furthermore, the phenotypes of the quadruply resistant mutants were those showing a higher resistance or a broader spectrum than expected by the addition of phenotypes of individual mutations. In addition, it was shown that, in contrast to the fact that hextuply resistant mutants could be isolated directly from the parent strain of M. tuberculosis, such mutants were not isolated directly from the parent B.C.G. strain, but could be isolated only after pre-incubation of the strain on a medium containing Tween 80.     

Chloramphenicol effects on DNA replication in UV-damaged bacteria

Increasing UV-doses to cultures of Escherichia coli strain B/r decreased progressively the amount of DNA which was formed in the presence of chloramphenicol (160 μg/ml) from the amount formed in unirradiated control cultures in chloramphenicol-containing medium. This is attributed to the progressive inactivation of active sites of DNA replication by UV. In order to form DNA the bacteria must then replicate from the chromosomal fixed origin, an activity which requires protein synthesis and thus cannot occur in the presence of chloramphenicol. Such damage was shown to be subject to photoreactivation after lower UV-doses and thus is the pyrimidine dimer. After higher doses non-photoreversible lesions began to accumulate so that all such damage became non-photoreversible after 96 erg/mm². The rate of synthesis of DNA in the presence of chloramphenicol was shown to be very close to the rate shown by bacteria incubated in the absence of chloramphenicol, indicating that all active sites of replication remaining after UV-damage remain active in the presence of chloramphenicol, as expected if the limiting effect of chloramphenicol is on initiation at the chromosomal origin and not due to reduction in rate of DNA replication.A much lower concentration of chloramphenicol (2 μg/ml) blocking only the chloramphenicol-sensitive event in control of DNA replication described by Ward and Glaser¹⁵, imposed a limitation in DNA accumulation in the culture of somewhat less than a doubling, as would be expected if the antibiotic at this concentration does not block the chloramphenicol-resistant control event. DNA degradation occured with incubation of bacteria given a UV-dose sufficient to inactivate all active DNA replication sites on their chromosomes, when in medium containing chloramphenicol concentrations (above 20 μg/ml) sufficient to block the chloramphenicol-resistant control event. Such breakdown resulted in death. The damage responsible for such death and DNA breakdown was not photoreversible after this dose, supporting the hypothesis that breakdown results from non-photoreversible inactivation of active DNA replication sites. This was in contrast to increased death in UV-damaged bacteria promoted by nalidixic acid, a specific inhibitor of DNA replication, which could be prevented in part by light exposure after the same UV-dose.     

The effect of hexose on chloramphenicol sensitivity and resistance in Chinese hamster cells.

The toxicity and extent of growth inhibition produced by chloramphenicol (CAP) in CAP^s Chinese hamster cells (line V79-5) was found to be dependent on the type and concentration of hexose in the medium. In high levels of glucose (6.5 mM), cultures of CAP^s cells underwent 7–8 population doublings in the presence of 100 μg/ml CAP and viability then dropped rapidly. In contrast, lower concentrations of glucose (1.0 mM) permitted only limited growth (2–3 doublings) in the presence of 100 μg/ml CAP, but the cells remained viable and apparently quiescent for prolonged periods of time. The growth potential of V79-5 cells in CAP appeared specifically dependent on glucose, as mannose and galactose could not substitute for glucose. The toxicity of CAP to these cells seemed to be determined primarily by the number of cell doublings in the presence of the drug. A CAP^R derivative of V79-5, designated 5-3, was analyzed in order to determine whether the requirement for glucose for cell growth in the presence of CAP also occurred in cells that were isolated as resistant to the drug. In order to rigorously control the hexose in the medium, some experiments were performed with medium containing dialysed, instead of whole, fetal calf serum. It was seen that the growth of the CAP^R line in the presence (but not the absence) of 100 μg/ml CAP was dependent on glucose in the medium. Thus, resistance to CAP in these cells appears to be a conditional state, dependent on glucose for expression. Furthermore, the glucose auxotrophy of these cells in the presence of CAP suggests that CAP is still affecting some activities in cells isolated as CAP^R.     

Cell division in Escherichia coli: evidence for regulation of septation by effector molecules

Evidence regarding the regulation of cell division has been obtained from the study of septation in a mutant of Escherichia coli. The mutant, MX74T2 ts52, gradually stops dividing when transferred from 30 to 41°C in rich medium, but forms long filaments and continues to synthesize DNA and protein. These filaments serve as test objects for the investigation of the regulation of septation. A synchronous cell division of the filaments is induced after 15 minutes, even at 41°C, by the addition of chloramphenicol (100 μg/ml.), rifampicin (200 μg/ml.), or by transfer to minimal medium. Blocking of protein formation with puromycin (500 μg/ml.) or amino-acid analogues does not permit septation. Thus, septation appears to be coupled to inhibition of peptide bond formation rather than protein synthesis. A model for the control of cell division is proposed in which a small effector molecule that is related to peptide bond formation is needed for septation.     

Mutational specificity of the dnaE173 mutator associated with a defect in the catalytic subunit of DNA polymerase III of Escherichia coli.

We developed a system to examine forward mutations that occurred in the rpsL gene of Escherichia coli placed on a multicopy plasmid. Using this system we determined the mutational specificity for a dnaE173 mutator strain in which the editing function of DNA polymerase III is impeded. The frequency of rpsL- mutations increased 32,000-fold, due to the dnaE173 mutator, and 87 independent rpsL- mutations in the mutator strain were analyzed by DNA sequencing, together with 100 mutants recovered from dnaE+ strain, as the control. While half the number of mutations that occurred in the wild-type strain were caused by insertion elements, no such mutations were recovered from the mutator strain. A novel class of mutation, named "sequence substitution" was present in mutants raised in the dnaE173 strain; seven sequence substitutions induced in the mutator strain occurred at six sites, and all were located in quasipalindromic sequences, carrying the GTG or CAC sequence at one or both endpoints. While other types of mutation were found in both strains, single-base frameshifts were the most frequent events in the mutator strain. Thus, the mutator effect on this class of mutation was 175,000-fold. A total of 95% of the single-base frameshifts in the mutator strain were additions, most of which occurred at runs of A or C bases so as to increase the number of identical residues. Base substitutions, the frequency of which was enhanced 25,000-fold by the mutator effect, occurred primarily at several hotspots in the mutator strain, whereas those induced in the wild-type strain were more randomly distributed throughout the rpsL sequence. The dnaE173 mutator also increased the frequency of duplications 28,000-fold. Of the three duplications recovered from the mutator strain, one was a simple duplication, the region of which was flanked by direct repeats. The other duplications were complex, one half part of which was in the inverted orientation of a region containing two sets of inverted repeats. The same duplications were also recovered from the wild-type strain. The present data suggest that dnaE173 is a novel class of mutator that sharply induces sequence-directed mutagenesis, yielding high frequencies of single base frameshifts, duplications with inversions, sequence substitutions and base substitutions at hotspots.     

Isolation and characterization of the symbiotic phenotype of antibiotic-resistant mutantsof Frankia from Rhamnaceae

Induced rifampicin-resistant mutants of Frankia were isolated by treatment of spores from strain ChI1 with 2 mg/ml of nitrosoguanidine. The mutagenic treatment was followed by growth for 72 h on non-selective medium to allow the expression of the mutation, before plating on selective medium. Spontaneous rifampicin-resistant mutants were isolated from strain ReI4 and chloramphenicol-resistant mutants were derived from strains ReI6 and TtI42. The mutants grew in medium containing up to 20 μg/ml of antibiotics. They showed similar morphology and growth pattern compared with their parent strains. However, they exhibited differences in symbiotic properties, such as infectivity and nitrogenase activity, from their parent strains.     

Biogenesis of mitochondria 34

Summary Mutants of the yeast Saccharomyces cerevisiae have been isolated in this laboratory which show increased resistance to a number of structurally and functionally unrelated antibiotics such as mikamycin, chloramphenicol, oligomycin and tetracycline (Bunn et al., 3971). When a multiply resistant haploid strain was crossed to an antibiotic sensitive strain, the resultant diploid progeny were completely resistant to chloramphenicol and oligomycin. However, the progeny showed different responses to mikamycin depending upon the concentration of antibiotic, all showed resistance to 25 g/ml but only about half were resistant to high levels of mikamycin (>100 g/ml). Detailed genetic analyses has shown that resistance to high levels of mikamycin is the result of a phenotypic interaction between two mutations, one nuclear and the other mitochondrial. The nuclear mutation by itself confers resistance to a number of antibiotics including chloramphenicol, oligomycin and mikamycin at a level of 25 g/ml. The mitochondrial mutation increases cellular resistance to mikamycin from 3 g/ml to about 8 g/ml. When the two mutations occur together in a cell, resistance to mikamycin is increased to at least 800 g/ml, the limit of solubility. Thus, the phenotypie interaction between these two mutations is not additive but synergistic.When cells containing the cytoplasmic [mik1-r] mutation are treated with ethidium bromide to produce ^° cells (no mtDNA), the [mik1-r] determinant is lost, indicating that this mutation is located in the mitochondrial DNA. Recombination analyses with other mitochondrial markers indicates a marker order of [oli1-r mik1-r ery1-r] with [mik1-r] showing tighter linkage to the [oli1-r] marker.     

Membrane protein synthesis in Escherichia coli: sensitivity to chloramphenicol

Analysis of the labeled proteins of Escherichia coli synthesized in the presence of chloramphenicol (30 or 100 μg/ml) showed: (A) no significant difference in the relative inhibitions of the envelope and cytoplasmic fractions but striking differences in the gel electrophoresis patterns of preparations from chloramphenicol-treated cells versus exponential phase cells; and (B) the average molecular weight of proteins labeled in chloramphenicol (100 μg/ml) was almost half the average of proteins from cells labeled in the absence of chloramphenicol, suggesting that they probably include some incomplete peptides. These results suggest that septation occurs in bacteria by assembly of preexisting envelope proteins in mutants that divide in the presence of high concentrations of chloramphenicol (100 μg/ml).     

Effect of prostaglandin producing modulators on in vitro growth of buffalo uterine epithelial cells

Studies were conducted to examine the effect of seven prostaglandin producing modulators on the in vitro growth of uterine epithelial cells in buffalo. The uterine epithelial cells isolated from slaughtered buffaloes were cultured in media containing a) Lipopolysaccaride (LPS): 0, 0.01, 0.1, 1, 10 and 100 μg/ml, b) linoleic acid: 0, 0.01, 0.1, 1, 10 and 100 μg/ml, c) linolenic acid: 0, 0.01, 0.1, 1, 10 and 100 μg/ml, d) oxytocin: 0, 10, 100, 1,000, 10,000 and 100,000 nm, e) tumor necrosis factor-α (TNF-α): 0, 0.05, 0.5, 1, 2.5 and 5 nm, f) progesterone: 0.1, 10, 25, 50, 75 and 100 nM, and g) estradiol: 0, 2.5, 5, 10, 20 and 50 nM. The control medium consisted of RPMI-1640 plus 10% bovine fetal serum. The growth of uterine epithelial were measured in terms of viability, cell number increment and monolayer formation. Results suggested that the growth of uterine epithelial cells were significantly (P < 0.05) higher in media containing 10 μg/ml, 10 μg/ml, 1 nm and 10 μg/ml linoleic acid, linolenic acid, TNF-α and LPS, respectively compared to control and lower doses used. Progesterone, estradiol and oxytocin did not significantly (P > 0.05) increase the growth of uterine epithelial cells. In conclusion, the growth of uterine epithelial cells increased when exposed to modulators in the order of linoleic acid ≥ linolenic acid ≥ LPS ≥ TNF-α > progesterone > estrogen > oxytocin.     

Mitochondrial activity of 2,6-diaminopurine in Saccharomyces cerevisiae.

Summary 2,6-diaminpurine (DAP) selectively inhibited mitochondrial protein synthesis in yeast cells with concomitant failure of cells to grow in non-fermentable (yeast extract, glycerol) medium. The selectivity was pronounced in all strains tested (15) nearly all of which were able to grow in yeast extract, glucose medium containing 5 mg/ml DAP (maximum solubility) whereas growth was arrested in all strains at 250–500 g/ml DAP in the glycerol medium. The inhibition was reversed by further addition of adenine to the culture medium. RNA synthesis in rat liver mitochondria was depressed by DAP suggesting that the analogue affected RNA polymerase activity.There was no evidence of nuclear mutagenicity by DAP but resistance to the antibiotics chloramphenicol and oligomycin was induced by the drug. Genetic evidence, although limited, indicated that the resistance mutations were cytoplasmic. The mitochondrial petite mutation was also induced by DAP but only at comparatively high concentrations. The mutagenic effects were seen only in the glycerol medium.     

Production of Corynecins by Chloramphenicol Resistant Mutants of Corynebacterium hydrocarboclastus

The isolation of chloramphenicol resistant strains from Corynebacterium hydrocarboclastus KY 4339 (rough type) was examined to seek a good source of corynecins (analogs of chloramphenicol). Various mutants resistant to chloramphenicol were isolated in the range from 50 to 1000 µg/ml by adaptation or induced mutagenesis by N-methyl-N′-nitro-N-nitro-soguanidine. Productivities of mutants related apparently to the degree of resistance from 50 to 500 µg/ml. Highly resistant mutants capable of growing in the presence of 1000 µg of chloramphenicol per ml showed decreased productivity which might be related to their lower growth rate in the fermentation medium.

Further attempts to derive resistant mutants to structural analogs of aromatic amino acids resulted in only a slight improvement of productivity, indicating that aromatic amino acids might play minor regulatory roles in corynecins synthesis.

The increase in productivity of corynecins by the best strain was about 4.5 fold of the parental strain.     

球形芽孢杆菌的抗药标记及外源DNA的转化与表达

用亚硝基胍（NTG）对球形芽孢杆菌（Bacillussphaericus）进行化学诱变,筛选到利福平（Rif）和链霉素（Sm）二个标记菌株。抗药浓度均达100u／ml培养基。其抗药性状能够获得较好地遗传。用含溶葡球菌酶基因的质粒DNA对RifR菌株进行原生质体转化,酶基因在该抗药菌株中获得了高效表达。经摇瓶发酵试验,溶葡球菌酶的活性约为122u／ml培养液。     

Induction of ethylene biosynthesis in tobacco leaf discs by cell wall disesting enzymes

Cellulysin induces ethylene production in tobacco leaf discs by initiating the formation of 1-aminocyclopropane-1-carboxylic acid. Induction occurred within 30 to 60 min of incubation and was inhibited by aminoethoxyvinylglycine, and the antiproteases, PMSF and soybean trypsin inhibitor. Cycloheximide (CHI) at 2.8 μg/ml and chloramphenicol (CAP) at 100 μg/ml did not inhibit this induction although incorporation of the label from (3,4-¹⁴C)methionine into the acid-insoluble fraction was inhibited by 57%. At 14 μg/ml CHI, and CAP, ethylene production was inhibited by 25% while protein synthesis was inhibited by 75%. We suggest that either the low amounts of protein synthesis that appear to be insensitive to CHI is sufficient to induce ethylene biosynthesis or that Cellulysin activates a preexisting but inactive form of ACC synthase to promote ethylene biosynthesis. Also, induction of ethylene production by microbial enzymes that digests plant cell walls may be an initial protective response of plants that serves to combat microbial infection.     

Clones of Chinese hamster cells cultivated in vitro not permanently resistant to azaguanine

The frequency of clones not permanently resistant to azaguanine (AG) was measured in Chinese hamster ovary cells (CHO) grown in vitro by plating them in 7.5 μg/ml AG and isolating a number of clones in the course of 5 experiments. Such isolated clones were propagated to a point at which their resistance to both AG and the reverse selective medium, HAT, could be determined. Out of a total of 13 clones isolated, 4 of these could not be distinguished from the parent CHO line, either on the basis of their growth in a gradient of AG concentrations or the reverse selective HAT medium or on the basis of their mutation frequency to resistance to 30 μg/ml AG. All four of the apparent phenocopies were isolated from plates in which although lower numbers of cells were seeded, a higher frequency of clones able to grow in AG was yielded. This suggests that the higher “mutation” frequencies obtained at lower cell densities are due to the appearance of phenocopies which occur only under these conditions. It is concluded that under low plating density conditions, the lower levels of AG (7.5 μg/ml) are not satisfactory for mutagenesis and mutation rate studies.     

Mutagenesis in cultured human diploid cells. III. Induction of 8-azaguanine-resistant mutations by furylfuramide.

Trans-2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (furylfuramide: FF or AF2) was tested for ability to induce 8-azaguanine (8AG) resistant mutations in cultured human diploid cells. FF had a relatively severe cytotoxic effect on the cells. From the concentration-survival curve, the D0 value for 2-h treatment with FF was estimated to be 11 mug/ml. When cells were treated with FF at various concentrations for 2 h, cultured in normal medium for 48 h, and then selected in medium containing 8AG at 30 mug/ml, the induced mutation frequency increased gradually with increase in concentration of FF. When cells were treated with FF at 10 mug/ml for 2 h, cultured in normal medium for various periods of mutation expression time, and selected with 8AG at 30 mug/ml, the highest induced mutation frequency was obtained with 48 h of mutation expression time. Microscopic examination of the numbers of cells in colonies indicated that the total number of cells increased by half during this mutation expression time of 48 h.     

Transductional construction of a threonine-hyperproducing strain of Serratia marcescens: lack of feedback controls of three aspartokinases and two homoserine dehydrogenases.

To construct a threonine-hyperproducing strain of Serratia marcescens Sr41, the six regulatory mutations for three aspartokinases and two homoserine dehydrogenases were combined in a single strain by three transductional crosses. The constructed strain, T-1026, carried the lysC1 mutation leading to lack of feedback inhibition and repression of aspartokinase III, the thrA1(1) mutation desensitizing aspartokinase I to feedback inhibition, the thrA2(1) mutation releasing feedback inhibition of homoserine dehydrogenase I, the two hnr mutations derepressing aspartokinase I and homoserine dehydrogenase I, and the etr-1 mutation derepressing aspartokinase II and homoserine dehydrogenase II. The strain produced ca. 40 mg of threonine per ml of medium containing sucrose and urea. Furthermore, the productivity of strain T-1026 was compared with those of strains devoid of more than one of the six regulatory mutations.     

MutS deficiency and activity of the error-prone DNA polymerase IV are crucial for determining mucA as the main target for mucoid conversion in Pseudomonas aeruginosa

Pseudomonas aeruginosa colonizes the respiratory tract of cystic fibrosis (CF) patients, where mutators along with mucoid variants emerge leading to chronic infection. Mucoid conversion generally involves mutations inactivating the mucA gene. This study correlates the frequency and nature of mucA mutations with the activity of factors determining the mutation rate, such as MutS and polymerase IV (Pol IV). Results show that: (i) the emergence frequency of mucoid variants was higher in isolates arising from mutS populations compared with the wild-type strain; (ii) in both strains mucoid conversion occurred mainly by mucA mutations; (iii) however, the mutator strain harboured mostly mucA22 (a common allele in CF isolates), while the wild type showed a wider spectrum of mucA mutations with low incidence of mucA22; (iv) disruption of dinB in the wild-type and mutS strains decreased drastically the emergence frequency of mucoid variants; (v) furthermore, the incidence of mucA mutations diminished in the mutS dinB double mutant strain which consisted only in mucA22; (vi) finally, the mucoid isolates obtained from the dinB strain showed an unexpected absence of mucA mutations. Taken together results demonstrate the implication of both MutS and Pol IV in determining mucA as the main target for conversion to mucoidy.