Evidence for a Relationship Between Deoxyribonucleic Acid Metabolism and Septum Formation in Escherichia coli

Septum formation is a key step in bacterial division, but the mechanism which controls periodic septum formation is unknown. In an attempt to understand this mechanism, lon(-) mutants, in which septum formation is blocked by very doses of ultraviolet light (UV), were investigated. UV must act on some part of the apparatus of cytokinesis; thus, identification of the UV target would identify part of this apparatus. As likely possibilities, UV might damage the septum-forming site or it might damage deoxyribonucleic acid (DNA), since DNA replication is normally coordinated with septum formation. To distinguish between these possibilities, DNA was specifically sensitized by incorporating bromodeoxyuridine into lon(-) bacteria. These bacteria were strongly sensitized to longer wavelength UV (2,900 to 3,100 A) so that they failed to form septa, grew into filaments which lysed, and did not form colonies. Various control experiments supported the conclusion that UV inhibits septum formation as a result of alterations in DNA metabolism. A relationship thus exists between DNA metabolism and septum formation.     

Analysis of the Escherichia coli Alp phenotype: heat shock induction in ssrA mutants

The major phenotypes of lon mutations, UV sensitivity and overproduction of capsule, are due to the stabilization of two substrates, SulA and RcsA. Inactivation of transfer mRNA (tmRNA) (encoded by ssrA), coupled with a multicopy kanamycin resistance determinant, suppressed both lon phenotypes and restored the rapid degradation of SulA. This novel protease activity was named Alp but was never identified further. We report here the identification, mapping, and characterization of a chromosomal mutation, faa (for function affecting Alp), that leads to full suppression of a Deltalon ssrA::cat host and thus bypasses the requirement for multicopy Kan(r); faa and ssrA mutants are additive in their ability to suppress lon mutants. The faa mutation was mapped to the C terminus of dnaJ(G232); dnaJ null mutants have similar effects. The identification of a lon suppressor in dnaJ suggested the possible involvement of heat shock. We find that ssrA mutants alone significantly induce the heat shock response. The suppression of UV sensitivity, both in the original Alp strain and in faa mutants, is reversed by mutations in clpY, encoding a subunit of the heat shock-induced ClpYQ protease that is known to degrade SulA. However, capsule synthesis is not restored by clpY mutants, probably because less RcsA accumulates in the Alp strain and because the RcsA that does accumulate is inactive. Both ssrA effects are partially relieved by ssrA derivatives encoding protease-resistant tags, implicating ribosome stalling as the primary defect. Thus, ssrA and faa each suppress two lon mutant phenotypes but by somewhat different mechanisms, with heat shock induction playing a major role.     

Second-site mutations in capR (lon) strains of Escherichia coli K-12 that prevent radiation sensitivity and allow bacteriophage lambda to lysogenize.

capR (lon) mutants of Escherichia coli K-12 are mucoid and sensitive to ultraviolet (UV) and X-ray radiation as well as to nitrofurantoin. The mutants form filaments after exposure to these agents. capR mutants are also conditionally lethal since they die when plated on complex medium even without UV treatment; this phenomenon is designated "complex medium-induced killing". Furthermore, capR mutants are poorly lysogenized by bacteriophage lambda. Second-site revertants were isolated by plating on media containing nitrofurantoin. All 17 of the independent revertants studied were still mucoid but resistant to UV radiation. Sixteen of the 17 revertants contained a mutation, sulA, that cotransduced with pyrD (21 min). A second locus, sulB, was also found that cotransduced with leu (2 min). Studies with partial diploids (F'pyrD+ sulA+/pyrD36 sulA17 capR9 (lon) demonstrated that sulA+ is dominant to sulA; thus the indicated partial diploid is UV sensitive, whereas the haploid parent is UV resistant. Furthermore, two other phenotypic traits of capR (lon) mutants were reversed by the sul mutation:complex medium-induced killing and the inability of lambda phage to efficiently lysogenize capR strains. On the basis of these and other results, the following model is suggested to explain capR (lon) and sul gene interactions. capR (lon) is a regulator gene for the structural genes sulA+ and sulB+. Depression of both sul operons results in UV sensitivity and decreased ability of lambda to lysogenize, whereas inactivation of either sul+ protein by mutation to sul prevents these phenomena.     

Isolation and Characterization of an Escherichia coli ruv Mutant Which Forms Nonseptate Filaments After Low Doses of Ultraviolet Light Irradiation

Two ultraviolet light (UV)-sensitive mutants have been isolated from Escherichia coli K-12. These mutants, designated RuvA(-) and RuvB(-), were controlled by a gene located close to the his gene on the chromosome map. They were sensitive to UV (10- to 20-fold increase) and slightly sensitive to gamma rays (3-fold increase). Host cell reactivation, UV reactivation and genetic recombination were normal in these mutants. Irradiation of the mutants with UV resulted in the production of single-strand breaks in deoxyribonucleic acid, which was repaired upon incubation in a growth medium. After UV irradiation, these mutants resumed deoxyribonucleic acid synthesis at a normal rate, as did the parent wild-type bacteria, and formed nonseptate, multinucleate filaments. From these results we concluded that the mutants have some defect in cell division after low doses of UV irradiation, similar to the lon(-) or fil(+) mutant of E. coli. The ruv locus was divided further into ruvA and ruvB with respect to nalidixic acid sensitivity and the effect of minimal agar or pantoyl lactone on survival of the UV-irradiated cell. The ruvB(-)mutant was more sensitive to nalidixic acid than were ruvA(-) and the parent strain. There was a great increase in the surviving fraction of the UV-irradiated ruvB(-) mutant when it was plated on minimal agar or L agar containing pantoyl lactone. No such increase in survival was observed in the ruvA(-) mutant.     

Genetic and Molecular Characteristics of X-Ray-Sensitive Mutants of Escherichia coli Defective in Repair Synthesis

Alleles responsible for X-ray-sensitive characteristics of three mutants of Escherichia coli B, which were also sensitive to ultraviolet (UV) irradiation, were mapped near metE locus, and named res-1, res-2, and res-3. All the res(-) mutants showed no host cell reactivability (Hcr(-)) for transducing deoxyribonucleic acid (DNA) of P1 phage irradiated by UV but they were Hcr(+) for infective DNA of P1 phage. Furthermore, they showed no detectable activity of DNA polymerase. Characteristics of allele res-1 were studied in detail. The mutant res-1 uvr(+) showed an extensive degradation of DNA after UV irradiation. Double mutants carrying res-1 uvrA(-), res-1 uvrB(-), and res-1 uvrC(-) showed no marked increase in UV sensitivity beyond that of the uvr(-) single mutants and only negligible UV-induced DNA degradation. The uvr(-) mutations showed no such suppressive effect on DNA degradation induced by X rays in these double mutants. It is concluded that res(-) mutants are defective in the second step (repair synthesis) of the excision repair process and that DNA polymerase is partly responsible for the assumed resynthesis step.     

Deoxyribonucleic Acid Synthesis and Cell Division in a lon− Mutant of Escherichia coli

The lon(-) mutants of Escherichia coli form long filamentous cells after temporary inhibition of deoxyribonucleic acid (DNA) synthesis by ultraviolet irradiation, treatment with nalidixic acid, or thymine starvation. The kinetics of DNA synthesis and cell division after a period of thymine starvation have been compared in lon(+) and lon(-) cells. After this treatment, both kinds of cells recover their normal DNA to mass ratio with the same kinetics. In contrast to previous reports, cell division is found to recommence in both lon(+) and in lon(-) cells after such a temporary period of inhibition of DNA synthesis. However, the delay separating the recommencement of DNA synthesis and of cell division is approximately three times as long in lon(-) as in lon(+) cells. Low concentrations of penicillin inhibit cell division in both lon(+) and lon(-) cells. In this case, cell division recommences with the same kinetics in both strains after the removal of penicillin. This suggests that different steps in the cell division process are blocked by inhibition of DNA synthesis and by penicillin treatment. The lon(-) mutation appears to affect the former of these steps.     

Overproduction of FtsZ suppresses sensitivity of lon mutants to division inhibition.

Escherichia coli lon mutants are sensitive to UV light and other DNA-damaging agents. This sensitivity is due to the loss of the lon-encoded ATP-dependent proteolytic activity which results in increased stability of the cell division inhibitor SulA. Introduction of the multicopy plasmid pZAQ containing the ftsZ gene, which is known to increase the level of FtsZ, suppressed the sensitivity of lon mutants to the DNA-damaging agents UV and nitrofurantoin. Alterations of pZAQ which reduced the expression of ftsZ reduced the ability of this plasmid to suppress the UV sensitivity. Examination of the kinetics of cell division revealed that pZAQ did not suppress the transient filamentation seen after exposure to UV, but did suppress the long-term inhibition that is normally observed. lon strains carrying pZAQ could stably maintain a multicopy plasmid carrying sulA (pBS2), which cannot otherwise be introduced into lon mutants. In addition, the increased temperature sensitivity of lexA(Ts) strains containing pBS2 was suppressed by pZAQ. These results suggest that SulA inhibits cell division by inhibiting FtsZ and that this interaction is stoichiometric.     

Role of the systems of DNA dark repair in determining bacterial and phage sensitivity to ultraviolet radiation in an ecological long-wave range]

A comparison has been made of sensitivity to far (254 nm), middle (300--315 nm) and near (315--400 nm) UV radiation of 12 strains of E. coli and 2 strains of B. subtilis differing in DNA dark repair (DR) capability. The mechanisms controlled by uvrA, uvrB, polA, recA, lon, and lexA genes are very effective in cells, irradiated by far and middle UV, but by 15--70% less effective in those irradiated by near UV. As the unirradiated bacteria poorly repair the near UV damaged phages (T7, lambda, SPPI), the low bacterial DR level after UV irradiation seems to be due to the unrepairable photoproduct formation in DNA.     

HtrA is a major virulence determinant of Bacillus anthracis

We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1(+) , pXO2(+) ), or in the ΔVollum (pXO1(-), pXO2(-), nontoxinogenic and noncapsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The ΔhtrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation-associated neutral protease A (NprA). VollumΔhtrA bacteria exhibit delayed proliferation in a macrophage infection assay, and despite their ability to synthesize the major B. anthracis toxins LT (lethal toxin) and ET (oedema toxin) as well as the capsule, show a decrease of over six orders of magnitude in virulence (lethal dose 50% = 3 × 10(8) spores, in the guinea pig model of anthrax), as compared with the parental wild-type strain. This unprecedented extent of loss of virulence in B. anthracis, as a consequence of deletion of a single gene, as well as all other phenotypic defects associated with htrA mutation, are restored in their corresponding trans-complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the ΔhtrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum ΔhtrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.     

提高产抗生素链霉菌紫外诱变正变率的研究

将UV诱变了的产抗生素链霉菌（Streptomyces sp.）AP 19-1菌株之孢子,置于适宜的生长温度27℃与接近抑制生长的胁迫温度33℃下培养,结果表明：在33℃下生长获得的子代菌株中,产抗生素水平超过其出发菌株的正向突变体所占的比例,明显比在27℃下培养的高。27℃下培养,正向突变体占总子代菌株数的25.8%,而在33℃下培养则为58.1%。用17种随机引物对出发菌株与UV诱变子代菌株进行总DNA的RAPD测验证明,在接近抑制生长的温度33℃下培养获得的子代,发生在其DNA水平上的变异程度比在27℃的要高得多。这一方法能较大幅度提高链霉菌紫外诱变育种的工作效率,同时也为链霉菌经紫外诱变后突变形成机制的进一步研究提供了新途径。Abstract: UV irradiated spores of Streptomyces sp. AP 19 -1 strain that can produce antibiotics were incubated at 27 ℃, and 33 ℃ which is close to inhibiting growth temperature, respectively. The results showed that there were much more forward mutants, whose level of producing antibiotics is higher than that of original strain, among the offspring of UV irradiated spores grown at 33 ℃, compared to that grown at 27 ℃. The percentage of the forward mutants was 25.8 % at 27 ℃ and 58.1% at 33 ℃. The progeny strains and the original strain were tested by RAPD using total DNA with 17 primers. It was demonstrated that more variations occurred in the chromosomal DNA of the progeny strains grown at 33 ℃ than in that at 27 ℃. This method facilitates increasing the efficiency of induced mutagenesis in breeding and provides a new way to study the mechanisms of mutation formation in UV irradiated Streptomyces sp. cells.     

The role of the (6-4) photoproduct in ultraviolet light-induced transition mutations in E. coli

Available evidence rules out the possibility that cyclobutane dimers are the major premutagenic lesions responsible for point mutations at sites of adjacent pyrimidine residues in the experiment systems examined to date in sufficient detail, that is, UV-induced mutations in chromosome loci in E. coli and UV-induced mutations in the cI gene of phage lambda. However, it is likely that the major cytotoxic effects of UV irradiation can be attributed to the cyclobutane pyrimidine dimer, as these lesions occur at 10 times the frequency of other UV-induced photoproducts in the dose range of 0.1-100 J/m2. The evidence also suggests that cyclobutane pyrimidine dimers are the major lesions responsible for induction of the SOS response and that as such they play an important, though indirect role, in the formation of mutations in irradiated DNA. Cyclobutane dimers may also be the major lesions responsible for other types of UV-light-induced mutations such as deletions. None of the available evidence rules out (6-4) photoproducts as a major premutagenic lesion induced by UV irradiation using these experimental systems. On the contrary, the mutation spectrum induced both in the lacI gene and the cI gene of phage lambda is that predicted for mutations induced by (6-4) photoproducts. The observation that neither the premutagenic lesions nor the (6-4) photoproduct is subject to enzymatic photoreactivation also implies that the (6-4) photoproducts are premutagenic. As reviewed above, neither the photosensitization experiments nor the action spectrum of the (6-4) photoproducts rules out such a role. Might a lesion other than the (6-4) photoproduct be the major premutagenic lesion responsible for point mutations in these experimental systems? It cannot be ruled out that another as yet undefined minor photoproduct that occurs with the same sequence distribution specificity as that of the (6-4) photoproduct and that is also not subject to the reactivating treatments is more mutagenic than the (6-4) photoproduct itself. Candidates for such a lesion might include a photohydrate of the (6-4) photoproduct itself or as yet undefined photoproducts. However, we believe these alternative possibilities to be remote.(ABSTRACT TRUNCATED AT 400 WORDS)     

Escherichia coli ras locus: its involvement in radiation repair

There are several classes of Escherichia coli mutants defective in radiation repair. These include strains defective in pyrimidine dimer excision, in photoreactivation, in recombination, in repair of X-ray damage, and ultraviolet (UV)-conditional mutants which do not divide after UV. Another mutant (ras(-)) has been isolated. The ras(-) has increased UV sensitivity, but only slightly increased X-ray sensitivity (1.5-fold increase). Ability to effect genetic recombination, to reactivate irradiated bacteriophage T1, and to be photoreactivated is normal. UV-induced mutation frequency is greatly increased in the mutant. The ras(-) apparently lacks the ability to repair some UV damage in the bacterial cell but can repair UV damage to bacteriophage DNA. The ras locus is located between lac and purE on the chromosome map.     

Plasmid mutations affecting self-maintenance and host growth in Escherichia coli.

As reported in the accompanying paper, a number of mutants of the ColVBtrp plasmid that can not be maintained stably in the host cell of Escherichia coli have been isolated. Each of the mutated plasmids has been transferred to an isogenic Col minus strain, and the resulting Col+ strains were studied to examine the effects of plasmid mutations on some properties of the host bacteria. Many of the strains harboring a mutated plasmid were thus found to be temperature sensitive; they failed to grow and divide normally at high temperatures. Some of them formed "filaments" under these conditions. These abnormal growth characteristics were accompanied by an increased susceptibility to sodium deoxycholate and methylene blue, suggesting that the cytoplasmic membrane has been altered. Moreover, studies of temperature-independent revertants obtained from two of these temperature-sensitive Col+ strains suggested that a single mutation on the plasmid is responsible for the pleiotropic effects exerted on the host cell. The bearing of these findings on the mode of replication and segregated of stringent-type plasmids such as ColVBtrp in the host bacteria is discussed.     

Genetic analysis of lon mutants of strain K-12 of Escherichia coli

Summary Following UV irradiation of AB1157 31 mucoid ultraviolet light UV sensitive mutants were isolated. These were all induced to form filaments by UV irradiation, i.e. they had all the phenotypic properties of Lon mutants. These lon mutants fell into two phenotypic classes based on their sensitivity to UV. The gene determining UV sensitivity and mucoidy in all mutants of both Class A and Class B was cotransducible with proC. Intra-class crosses by Pl transduction yielded no UV resistant recombinants. Inter-class crosses yielded UV resistant nonmucoid recombinants, the frequency depending on the direction of the cross. The data imply two adjacent blocks in the lon region of E. coli and the order of markers in this region is probably proC tsx lon Class A lon purE Class B.This work was carried out under Public Health Service Grant CA 05687-08 from the National Cancer Institute.Recipient of a Public Health Service Career Development Award.