Isolation of a Serratia marcescens mutant which is an efficient recipient for the E. coli episome

Summary A Serratia marcescens mutant, which is an efficient recipient for the Escherichia coli episome (F' plasmid), was isolated after mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine (NTG). Episomes could be maintained in the mutant cell under conditions selective for a gene on the plasmid. This S. marcescens mutant could also be transformed with pBR 322 DNA at a frequency higher than that of the parental strain.     

Temperature-dependent and amber copy mutants of plasmid R1drd-19 in Escherichia coli.

The isolation of conditional mutants with an altered copy number of the R plasmid R1drd-19 is described. Temperature-dependent as well as amber-suppressible mutants were found. These mutant plasmids have been named pKN301 and pKN303, respectively. Both types of mutations reside on the R plasmid. No difference in molecular weight could be detected by neutral sucrose gradient centrifugation for any of the mutant plasmids when compared with the wild-type plasmid. The number of copies of the plasmids was determined by measurement of the specific activity of the R plasmid-mediated β-lactamase and by measurement of covalently closed circular (CCC) DNA in alkaline sucrose gradients and dye-CsCl density gradients. Below 34 °C the temperature-dependent mutant, pKN301, had the same copy number as the wild type, while this was four times that of the wild type above 37 °C. The amber mutant pKN303 had a copy number indistinguishable from that of the wild-type plasmid in a strain containing a strong amber suppressor and a copy number about five times that of the wild-type plasmid in a strain lacking an amber suppressor. In a strain containing a temperature-sensitive amber suppressor, the amber mutant's copy number increased with the decrease in amber suppressor activity. Thus, the existence of the temperature-dependent and the amber-suppressible R-plasmid copy mutants indicates that the system that controls the replication of plasmid R1drd-19 contains an element with a negative function and that this element is a protein.     

Biochemical Studies of Two Bacillus pumilus Plasmids

Bacillus pumilus NRS 576 harbored an estimated two copies per chromosome of a covalently closed, circular (CCC) deoxyribonucleic acid (DNA) molecule, the 576 plasmid. The 576 plasmid has a buoyant density of 1.698 g/cm(3) and a molecular weight of about 28 x 10(6). Plasmid copy number remained about the same in both exponentially growing and stationary-phase cells. Spontaneous variants of NRS 576 that formed spores at an elevated frequency were designated as W mutants. W mutants appeared to have lost the 576 plasmid on the basis of the following: W mutants (38 tested) lacked detectable CCC DNA, and the majority of the plasmid homologous sequences in bulk NRS 576 DNA were absent from bulk W mutant DNA. B. pumilus ATCC 7065 harbored at least 10 copies per chromosome of a CCC DNA element, the 7065 plasmid. The 7065 plasmid has a buoyant density of 1.696 g/cm(3) and a molecular weight of about 6 x 10(6). Although the copy number of the plasmid appeared to remain the same in exponentially growing and stationary-phase cells, an additional CCC form of higher molecular weight was detected in stationary-phase cells.     

Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation.

A method that gives high recovery of deoxyribonucleic acid (DNA) from crude bacterial lysates using ethidium bromide-cesium chloride density gradient centrifugation is presented. After Pronase digestion and shearing of the lysate, essentially 100% recovery of chromosomal DNA and a reproducible recovery of covalently closed circular (CCC) plasmid DNA is obtained for a specific plasmid in a given strain. This method should be useful for comparing the CCC plasmid/chromosome ratio of various plasmid-host combinations.     

Plasmid-mediated control of nodulation in Rhizobium trifolii

The nodulation ability was effectively eliminated from different Rhizobium trifolii strains incubated at elevated temperature (urkowski and Lorkiewicz, 1978). Non-nodulating (Nod^-) mutants were stable and no reversion of Nod^- to Nod⁺ phenotype was observed. Strains R. trifolii 24 and T12 which showed a high percentage of elimination of nodulation ability were examined in detail. Two plasmids were detected in strain 24 using neutral and alkaline sucrose gradient centrifugation of plasmid preparations. Molecular weights of the plasmids pWZ1 and pWZ2 were 460 Mdal and 190 Mdal, respectively. Rhizobium lysates labeled with ³H-thymidine and ultracentrifuged in caesium chloride — ethidium bromide gradients demonstrated a 40% reduction of the plasmid DNA content in R. trifolii 24 Nod^- mutants in comparison with the nodulating wild type strain 24. It was found further that non-nodulation of mutants 24 Nod^- was due to the absence of plasmid pWZ2. Sucrose gradient data also demonstrated that strain T12 contained two plasmids with molecular weights corresponding to those of pWZ1 and pWZ2, respectively. In Nod^- mutant clones derived from strain T12, pWZ2 plasmid was missing.Non Standard Abbreviations CCC covalently closed circular - OC open cirucular - Sarkosyl sodium N-lauroylsarcosinate     

Effect of mutagens, chemotherapeutic agents and defects in DNA repair genes on recombination in F' partial diploid Escherichia coli.

The ability of mutagenic agents, nonmutagenic substances and defects in DNA repair to alter the genotype of F' partial diploid (F30) Escherichia coli was determined. The frequency of auxotrophic mutants and histidine requiring (His-) haploid colonies was increased by mutagen treatment but Hfr colonies were not detected in F30 E. coli even with specific selection techniques. Genotype changes due to nonreciprocal recombination were determined by measuring the frequency of His- homogenotes, eg. F' hisC780, hisI+/hisC780, hisI+, arising from a His+ heterogenote, F' hisC780 hisI+/hisC+, his1903. At least 75% of the recombinants were homozygous for histidine alleles which were present on the F' plasmid (exogenote) of the parental hetergenote rather than for histidine alleles on the chromosome. Mutagens, chemotherapeutic agents which histidine alleles on the chromosome. Mutagens, chemotherapeutic agents which block DNA synthesis and a defective DNA polymerase I gene, polA1, were found to increase the frequency of nonreciprocal recombination. A defect in the ability to excise thymine dimers, uvrC34, did not increase spontaneous nonreciprocal recombination. However, UV irradiation but not methyl methanesulfonate (MMS) induced greater recombination in this excision-repair defective mutant than in DNA-repair-proficient strains. Mutagenic agents, with the exception of ethyl methanesulfonate (EMS), induced greater increases in recombination than the chemotherapeutic agents or the polA1 mutation. EMS, which causes relatively little degradation of DNA, was more mutagenic but less recombinogenic than MMS, a homologous compound ths that inhibition of DNA occurring single-stranded regions in replicative intermediates of the DNA. Mutagens which cause the rapid breakdown of DNA may, in addition, introduce lesions into the genome that increase the number of single-stranded regions thus inducing even higher frequencies of recombination.     

A novel type of E. coli mutants with increased chromosomal copy number

We have isolated E. coli mutants which can grow at 30 degrees C but not at 42 degrees C and are able to harbor the oriC plasmid (minichromosome) at a higher copy number than the parental wild-type strain at the permissive temperature. The mutants were found to contain higher amounts of chromosomal DNA per mg protein than the wild-type, whether or not they harbor the plasmid. Experimental results suggest that the higher amount of chromosomal DNA is due to a higher copy number of chromosomes and not to a larger amount of DNA per chromosome. These properties in each of the mutants are caused by a single mutation at the rpoB or rpoC gene that code for the beta or beta' subunit of RNA polymerase, respectively. The mutations are thought to affect the regulation of replication of oriC-bearing replicons, that is, the E. coli chromosome and oriC plasmids, but not the miniF plasmid.     

Evidence for plasmid-associated crystal toxin production in Bacillus thuringiensis subsp. israelensis

Three crystalliferous (Cry⁺) strains of Bacillus thuringiensis subsp. israelensis (serotype 14) that produce parasporal protein crystals toxic to dipteran larvae and several acrystalliferous (Cry^?) mutants, either induced or spontaneously derived from a single Cry⁺ parent, were examined for the presence of covalently closed circular (CCC) DNA in attempts to correlate toxin production with the presence of a specific plasmid. The plasmid profiles of both Cry⁺ and Cry^? variants were analyzed by both a cleared lysate- and a modified Eckhardt lysateelectrophoresis technique. All of the Cry^? mutants derived from the Cry⁺ parental strain had lost a 4.0- to 4.4-megadalton (Mdal) plasmid. Bioassay data confirmed loss of toxin production by the Cry^? variants. All three Cry⁺ strains, including the parent of the Cry^? strains, contained CCC plasmids DNAs of the following approximate molecular weights: 4.0 to 4.4, 5.2 to 6.0, and 11.4 to 13.0 Mdal. One Cry⁺ strain contained an additional CCC plasmid of 6.7 to 7.2 Mdal. The plasmid patterns for several Cry^? derivatives differed in other respects from the pattern for their parent strain. The various Cry⁺ and Cry^? strains could be distinguished either by phenotypical differences in antibiotic sensitivity, crystal production, and toxicity, or by differences in their plasmid profiles.     

Study of the regulation of crp gene expression in Escherichia coli K12

The regulation of crp gene expression by CRP-cAMP complex was studied in E. coli strain by the crp-lac operon fusion. F'141 crp+ episome decreased 5-7 fold the high level of crp-lac expression in crp strains while F'141 crp episome had no effect. The hybrid plasmid pCAP2 crp+ with the intact crp gene did not affect the crp gene expression level in crp mutants, though they had acquired the Crp+ phenotype just as they did in F'141 crp+ presence. The F'141 crp+ and pCAP2 crp+ combination in crp mutants also resulted in decrease of the crp gene expression comparable to the registered in the presence of the F'141 crp+ plasmid. Similar repression occurred only in cya+ strains but not in cya strains. The crp gene is supposed to possess negative regulation by CRP-cAMP complex with a complementary factor also necessary. The latter is evidently located in an E. coli chromosome site overlapped by F'141 episome.     

A gene affecting accumulation of the RNA moiety of the processing enzyme RNase P.

The level of 10Sb (M1) RNA, the RNA of RNase P, is very low in growing cultures of rnpB mutants. Northern transfer experiments suggested that these strains accumulate no more than 10% of the wild-type level of 10Sb RNA. However, there is no indication that there is a limiting amount of RNase P activity in these mutants in vivo. A plasmid that directs the synthesis of 10Sb RNA does not complement the rnpB mutants, even though there is only a single gene for 10Sb RNA in the Escherichia coli genome. The 10Sb RNA synthesized from this plasmid is equivalent to wild-type 10Sb RNA since it can replace it in the reconstitution of RNase P. The 10Sb RNA, which is a rather stable molecule, is unstable in the presence of the rnpB mutation. This could explain why rnpB mutants do not accumulate 10Sb RNA. An F' plasmid that contains DNA from the rnpB region of the chromosome complements an rnpB mutant in vivo and in vitro, and it also contains the 10Sb RNA gene. A number of possible explanations for these phenomena are discussed.     

micF RNA in ompB mutants of Escherichia coli: different pathways regulate micF RNA levels in response to osmolarity and temperature change.

The repressor RNA, micF RNA, is regulated by temperature, osmolarity, and other stress conditions during growth of Escherichia coli. Northern (RNA) blot analyses showed that levels of micF RNA differ widely in various ompB mutant strains when cells are grown at 24 degrees C in LB broth. For example, relative to the parental strain MC4100, the ompR101 mutant strain (which contains no functional OmpR) had about a 10-fold reduction in micF RNA, whereas the envZ11 strain showed about a 5-fold increase. At 37 degrees C, however, micF RNA levels in the ompR101 and envZ11 strains and other ompB mutants differed by less than two-fold compared with the level in strain MC4100, thus indicating that a factor(s) independent of the ompB locus regulates micF RNA expression with temperature increase and that there is an additional control mechanism(s) which maintains the levels of micF RNA in these mutants close to that of the wild type during growth at high temperatures. In a plasmid strain containing the micF gene but without the upstream OmpR-binding site, steady-state levels of micF RNA increased with temperature increase but did not change with osmolarity increase. This showed that osmolal regulation but not temperature regulation of micF depends on these upstream sequences and suggested that while osmolal regulation of the micF gene depends on OmpR, thermal regulation does not.     

Genetic analysis of a temperature-sensitive Salmonella typhimurium rho mutant with an altered rho-associated polycytidylate-dependent adenosine triphosphatase activity.

A conditional-lethal rho mutant of Salmonella typhimurium LT2 has been isolated. The mutation was selected as a suppressor of the polarity of an insertion sequence (IS)2-induced mutation (gal3) carried on an F' plasmid. In addition to suppression of IS2-induced polarity, the rho-111 mutation suppressed nonsense and frameshift polarity. The rho-associated polycytidylic acid-dependent adenosine triphosphatase activity in the mutant strain was elevated 15-fold above that in the parental strain, and the mutant rho protein was thermally unstable. A temperature-resistant revertant of the mutant strain did not suppress polarity and contained normal levels of polycytidylic acid-dependent adenosine triphosphatase, suggesting that the phenotype of the rho-111-bearing strain is the consequence of a single mutation. The rho-111 mutation was located on the S. typhimurium linkage map midway between the ilv and cya loci by phage P22 cotransduction studies. F' plasmid maintenance was not impaired in the mutant strain, and the mutation was recessive to the wild-type allele. The rho-111 mutation did not alter in vivo expression of either the tryptophan or histidine operons.     

Expression of a mutation affecting F incompatibility in the integrated but not the autonomous state of F.

Previously we have described a mutant Hfr strain in which incompatibility between the integrated F factor and an autonomous F-prime (F') factor was abolished. The mutation (inc) was located in the integrated F factor. F-prime factors isolated from the mutant Hfr strain have the same incompatibility behavior as those isolated from normal Hfr strains. Reintegration of these F' factors into the chromosome restores the Inc- phenotype characteristic of the mutant Hfr. The inc mutation thus affects incompatibility between integrated F and autonomous F(Fi-Fa incompatibility) but not incompatibility between two autonomous F factors (Fa-Fa incompatibility). The implications of this finding for the mechanism of plasmid incompatibility are discussed.     

marA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli.

Stable chromosomal multiple-antibiotic-resistant (Mar) mutants of Escherichia coli, derived by exposing susceptible cells to low concentrations of tetracycline or chloramphenicol, express cross-resistance to structurally unrelated antibiotics. The entire resistance phenotype is reversed to susceptibility by insertion of transposon Tn5 into a locus, designated marA, near 34 min on the chromosome (A. M. George and S. B. Levy, J. Bacteriol. 155:541-548, 1983). Strains in which 39 kbp of chromosomal DNA, including marA, had been deleted were unable to produce Mar mutants. The deletion strain could be complemented in trans by introduction of intact marA+ on plasmid F'506. Junction fragments from a strain containing marA::Tn5 were cloned, exploiting kanamycin resistance on Tn5 for selection. They were used as probes to search a phasmid library of E. coli K-12 for recombinants containing the marA+ region. Two phasmids which contained regions hybridizing to this probe were identified and shown to complement delta marA in a deletion strain. From one phasmid, several marA-containing fragments were cloned: those of greater than or equal to 7.8 kbp restored the ability to form Mar mutants in a deletion strain. These Mar mutants were shown to be dependent on the cloned marA fragment. Chromosomal as well as recombinant Mar mutants showed increased expression of a marA-specific mRNA species of about 1.4 kb, which was barely or not detectable in wild-type strains. Exposure of mutants and, to a lesser extent, parental strains to tetracycline or chloramphenicol resulted in elevated levels of mRNA which hybridized to the marA probe. These results indicate that the marA locus is needed for production of Mar mutants and is regulated, responding to at least two antibiotics to which it controls resistance.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain.

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Cloning of genes affecting capsule expression in Staphylococcus aureus strain M

Staphylococcus aureus strain M produces large amounts of capsular polysaccharide. It produces a non-encapsulated variant at a frequency of 0.01% at 37 degrees C. At high temperature (43 degrees C), the frequency of capsule loss was shown to be 1-38%. A 19 kb plasmid and a prophage were found to be carried by the M strain, but curing of these elements did not affect capsular production. To clone the capsular (cap) genes, a plasmid library of S. aureus M was constructed directly in S. aureus RN4200. The library was then infected with phage 80 alpha. After transduction of the phage lysates to a Cap- mutant derived from M strain, a recombinant plasmid was obtained which complemented the mutant to a Cap+ phenotype. Chromosomal walking experiments were used to clone additional nearby cap genes. Complementation tests using a collection of Cap- mutants showed that most of the mutants were complemented by a 19.4 kb DNA fragment, suggesting that the majority of the cap genes affecting capsule production are clustered together.     

Roles of chromosomal and episomal dinB genes encoding DNA pol IV in targeted and untargeted mutagenesis in Escherichia coli

DNA polymerase IV (pol IV) in Escherichia coli is a member of a novel family of DNA polymerases (the DinB/UmuC/Rad30/Rev1 super-family or the DNA polymerase Y family). Although expression of the dinB gene encoding DNA pol IV is known to result in an enhancement of untargeted mutagenesis, it remains uncertain whether DNA pol IV is involved in a variety of lesion-induced mutagenesis (targeted mutagenesis), and the relationship between expression levels of dinB and the mutagenesis that DNA pol IV promotes has not been investigated thoroughly. Here, we report that DNA pol IV is involved in -1 frameshift mutagenesis induced by 4-nitroquinoline N-oxide (4-NQO) and that the expression level of the chromosomal pol IV gene is 6-12 times higher than those for other SOS-inducible DNA polymerases in E. coli, i.e., DNA pol II (PolB) or DNA pol V (UmuDC), respectively. Interestingly, the dinB gene is present not only on the chromosome but also on the F' plasmid in the E. coli CC108 strain. In this strain, 750 molecules of DNA pol IV are expressed from the F' dinB gene in the uninduced state and 250 molecules are expressed from the chromosomal gene. These cellular expression levels strongly affect -1 frameshifts induced by 4-NQO in runs of six guanine bases: mutagenicity was highest in the strain CC108, followed by strains YG2242 (chromosome deltadinB/F' dinB+), YG2247 (chromosome dinB+/F' deltadinB) and FC1243 (chromosome deltadinB/F' deltadinB). The incidence of untargeted -1 frameshifts was reduced by two-thirds on deletion of dinB from the F' episome. The chromosomal dinB gene appeared to have little or no effect on the untargeted mutagenesis. These results suggest that DNA pol IV efficiently mediates targeted mutagenesis by 4-NQO, and that the cellular levels of expression substantially affect targeted and untargeted mutagenesis.     

Constitutive inhibition of DNA degradation due to the enzyme RecBCD in the radiation-resistant Escherichia coli K-12 mutant Gam(r)444]

Exonucleolytic degradation of [3]H-labeled DNA was examined in partially purified fractions of lysates obtained from nonirradiated RecBCD enzyme-containing cells of Escherichia coli and in the radiation-resistant mutant Gamr444. The degradative activity was shown to be lowered in these cells to the same extent as in the recBC mutant. The efficiency of plating of the mutant phage T4 2-, DNA of which can be degraded by exonuclease V, was 400-fold higher on the strain Gamr444 than on the wild-type strain AB1157. This value was shown to be only twice as low as that on the recB mutant or on the strain AB1157 carrying plasmid pGam26 with a radiation-resistance allele gam26 cloned from mutant Gamr444. The data obtained confirmed the hypothesis that the Gamr444 mutant contains a constitutive inhibitor of exonucleolytic activity of the RecBCD enzyme in nonirradiated cells. This inhibitor was shown to be encoded by the gam26 allele that had previously been mapped at 56.8 min of the E. coli chromosome. A possible mechanism of the involvement of this inhibitor in enhanced radiation resistance of the mutant Gamr444 is considered.