Behavior of Some Episomal Elements in a Recombination-Deficient Mutant of Escherichia coli

Conjugal transfer and autonomous replication of some episomes occurred normally in a recombination-deficient (Rec^–) mutant of Escherichia coli K-12. Transduction with phage Plbt of an R factor also occurred normally in this Rec^– mutant, but complete or abortive transduction with Plbt of chromosomal genes did not occur. In contrast, transduction of galactose genes by phage λ_dg occurred in the Rec^– bacteria as frequently as in the Rec⁺ strain. It was shown that phage Plbt does not grow at all on the Rec–bacteria. Recombination between two different R factors, two mutants of phage λ and two mutants of phage T4 occurred normally in the Rec^– bacteria, but did not give a Rec⁺ phenotype to the host bacteria. Colicinogenic factor I made the Rec^– host bacteria more resistant to ultraviolet light but the colicinogenic strain was still infertile in the crosses with the Hfr srains of E. coli K-12.     

Detection of transcribable recombination products following conjugation in rec+, reCB- and recC-strains of Escherichia coli K12

By crossing Hfr and F^? strains of Escherichia coli which carry non-identical (but non-complementing) lacZ^? mutations, the detection of β-galactosidase produced from LacZ⁺ recombination products is possible, beginning 60 minutes after transfer of the Hfr lac^? allele. This system was used to show that when the F^? cells carry recB^?, almost normal amounts of LacZ⁺ enzyme are formed even though the number of viable recombinants is less than 1% of the Rec⁺ level. A similar result is found when the F^? cells carry recC^?. In contrast, LacZ⁺ enzyme activity is not detected either when RecA^? F^? cells are used or in a stable RecA^? merodiploid carrying the two lacZ^? alleles.     

Translocation of antibiotic resistance markers of a plasmid-free Streptococcus pyogenes (group A) strain into different streptococcal hemolysin plasmids

Summary Wild-type strain A454 (Streptococcus pyogenes) transferred en bloc its erythromycin (Em) and tetracycline (Tc) resistance markers into several plasmid-free streptococcal recipients. No plasmid DNA was detected in either the wild-type or the transconjugant strains. Crosses were performed between A454 and S. faecalis Rec⁺ or Rec^- recipients carrying hemolysin-bacteriocin plasmids, pIP964 or pAD1. The Em Tc-resistant transconjugants obtained harbored either the parental plasmid or an Em Tc resistance plasmid derived from pIP964 or pAD1. The restriction endonuclease analysis of 12 derivative plasmids showed insertions of various sizes into different fragments of pIP964 or pAD1. A454 and the Em Tc-resistant plasmid-free transconjugants were found to contain two EcoRI DNA fragments, that shared homology with ³²P-labeled pIP1077, one of the Em Tc resistance derivative plasmids, but not with ³²P-labeled pIP964. No homology was detected between pIP1077 and the cellular DNA of the antibiotic-susceptible recipients.Previously Thea Horodniceanu     

The segregation of the SbcA and Rac phenotypes in an Escherichia coli recB mutant

Summary In certain HfrxF^– recB ^– crosses recombinant progeny were examined for their SbcA and Rac phenotypes. Recombinants which inherited either his ⁺ or trp ⁺ from the donor in an Hfr recB21 sbcA8xF^– recB21 Rac^–SbcA⁺ cross acquired the RecB⁺ phenotype in most instances (presumably by inheriting the sbcA8 allele). Several independent Rec⁺ (sbcA8) recombinants from this cross were converted back to the Rec^– (sbcA ⁺) phenotype by mating with a Rac⁺ SbcA⁺ Hfr. Ten out of 14 of these Rec^– recombinants retained the Rac^– phenotype of the original parent. It was concluded that these results were inconsistent with the hypothesis that sbcA is a gene carried by a Rac prophage.     

On the ability of Salmonella typhimurium cells to form deoxycytidine nucleotides.

Summary The fate of the donor DNA after conjugation in Escherichia coli was studied through crosses with a Hfr lacZ5 donor and several F^- lacZ22 recipients. The fate of the donor allele was studied by assaying the -galactosidase activity formed by complementation between the lacZ5 allele and the lacZ22 allele. We used continuous cultures of the recipient in order to be able to study the fate of the donor DNA during many generations under constant physiological conditions. We could show that the donor DNA allele is inactivated in Rec⁺, recA171 and recB21 recipient cells. The inactivation rate depends on the nature of the recipient, Rec⁺ or recombination deficient, and especially in the case of the recombination deficient mutants on the growth rate of the recipient.     

Early transfer of genes determining transfer functions by some Hfr strains in Escherichia coli K12

Summary Sixty-eight Hfr strains were examined for their ability to transfer early in conjugation the transfer genes carried by the integrated sex factor. This was measured by mating these strains with F^- phenocopied recipient cultures of strains carrying transfer-deficient Flac ⁺ factors, and then measuring the ability of the recipient strains to transfer lac ⁺ to a further recipient strain. Most Hfr strains did not complement the missing transfer functions, though in some strains complementation was observed. It is concluded that on the sex factors of different Hfr strains either the site at which integration occurs or the origin of transfer must vary.     

Does Tn10 transpose via the cointegrate molecule?

Summary It has been well established that Tn3 and its relatives transpose from one replicon to another by two successive reactions: formation of the cointegrate molecule and resolution from it. Whether or not the 9300 base pair tetracycline resistance transposon Tn10 transposes in the same manner as Tn3 was investigated by two methods.In the first method, 55, a lambda phage carrying Tn10 was lysogenized in an Escherichia coli strain carrying a Tn10 insertion; the phage has a deletion in attP, hence it was lysogenized in a Tn10 sequence in the E. coli chromosome by reciprocal recombination. The chromosomal structure in these lysogens is equivalent to the Tn10-mediated cointegrate molecule of lambda and the E. coli chromosomal DNA. The stability of the cointegrate molecule was examined by measuring the rate of excision of lambda from the host chromosome, and was found to be stable, especially in a Rec^- strain. Because of this stability, the cointegrate molecule should be accumulated if Tn10 transposes via the cointegrate molecule. Then, we examined the configuration of products made by transposition of Tn10 from 55 to the E. coli chromosome. The cointegrate molecule was found in products of Tn10 transposition in a Rec⁺ strain at a frequency of 5% per Tn10 transposition, but this molecule could not be found in a Rec^- strain. Since transposition of Tn10 was recA-independent, absence of the cointegrate molecule formed in a RecA^- strain strongly suggested that the cointegrate molecule is not an obligatory intermediate of transposition of Tn10.In the second method, mobilization of pACYC177 by R388 and by R388:: Tn10 was examined. The pACYC177 plasmid was mobilized by R388::Tn10 at a frequency of 10^-4 per donor but not by R388. It occurred, in most cases, by inverse transposition of R388::Tn10 to pACYC177 forming plasmids such as pACYC177::IS10-R388-IS10. Mobilization of pACYC177 by a Tn10-mediated cointegrate in the form of pACYC177::Tn10-R388-Tn10 was not observed in crosses using a Rec^- donor. These observations also suggested that transposition of Tn10 in Rec^- cells does not occur via the cointegrate molecule.     

Growth and nodulation competitiveness of Sinorhizobium meliloti L1 (RecA−) is less than that of its isogenic strain L33 (RecA+) but comparable to that of two S. meliloti wild-type isolates

Gnotobiotic systems were used to assess the competitive abilities of bioluminescent Sinorhizobium meliloti strains L1 (RecA⁻) and L33 (RecA⁺) for growth and host plant nodulation in the presence of a reconstructed S. meliloti population. Three wild-type strains belonging to infective subgroups of a natural S. meliloti population were chosen as competitors in microcosm studies. Whereas the RecA⁺ strain L33 dominated the reconstructed population with respect to growth and alfalfa nodulation, the competitiveness of the RecA⁻ strain L1 was reduced compared to that of one of the field strains, but comparable to that of the other field isolates. This result indicates that strain L1, despite its recA mutation, has the potential to compete successfully with a resident S. meliloti population after environmental release. Received: 4 November 1996 / Received revision: 9 January 1997 / Accepted: 17 January 1997     

ATP Production after ultraviolet irradiation inEscherichia coli

Doses of ultraviolet (UV) irradiation up to 40 Jm^–2 produce a continuous increase of both the level and the rate of ATP production in a RecA⁺ strain ofEscherichia coli growing in AB minimal medium with glucose as carbon source. At fluences higher than 40 Jm^–2, these two parameters increase at a constant rate independent of the dose. Furthermore, in the UV-irradiatedrecA13 mutant, the concentration of ATP is higher and its rate of production is faster than in the RecA⁺ strain. The same behavior as cited above was shown by both RecA⁺ and RecA^– cells when incubated in AB minimal medium without any carbon source both 60 min before and after UV irradiation.     

IS21 insertion in the trfA replication control gene of chromosomally integrated plasmid RP1: a property of stable Pseudomonas aeruginosa Hfr strains

Summary Broad host range IncP-1 plasmids are able to integrate into the chromosome of gram-negative bacteria. Strains carrying an integrated plasmid can be obtained when the markers of a temperature-sensitive (ts) plasmid derivative are selected at non-permissive temperature; in this way Hfr (high frequency) donor strains can be formed. The integrated plasmids, however, tend to be unstable in the absence of continuous selective pressure. In order to obtain stable Hfr donor strains of Pseudomonas aeruginosa PAO, we constructed a derivative of an RP1 (ts) plasmid, pME134, which was defective in the resolvase gene (tnpR) of transposon Tn801. Chromosomal integration of pME134 was selected in a recombination-deficient (rec-102) PAO strain at 43°C. Plasmid integration occurred at different sites resulting in a useful set of Hfr strains that transferred chromosomal markers unidirectionally. The tnpR and rec-102 mutations prevented plasmid excision from the chromosome. In several (but not all) Hfr strains that grew well and retained the integrated plasmid at temperatures below 43°C, the insertion element IS21 of RP1 was found to be inserted into the trfA locus (specifying an essential trans-acting replication funtion) of the integrated plasmid. One such Hfr strain was rendered rec ⁺; from its chromosome the pME134::IS21 plasmid (=pME14) was excised and transferred by conjugation to Escherichia coli where pME14 could replicate autonomously only when a helper plasmid provided the trfA ⁺ function in trans. Thus, it appears that trfA inactivation favours the stability of chromosomally integrated RP1 in P. aeruginosa.     

Some Genetic Consequences of Changes in the Level of recA Gene Function in ESCHERICHIA COLI K-12

Genetic recombination was studied in E. coli mutants that carry lesions in the recA gene but retain some capacity for generating recombinant progeny. We observed that recombination was detectable only at a very low level during the incubation of leaky RecA^- merozygotes in broth. However, recombination appeared to occur at much higher frequencies when recombinant progeny were assayed by selection on minimal agar. Analysis of the recombinants obtained with Hfr donors revealed a deficiency of multiple exchanges per unit length of DNA in leaky RecA ^- strains. In many of these crosses recombinants that inherited donor alleles close to the transfer origin were much reduced in frequency, except when the recipient was also RecB^-.     

Recombination deficient mutant of Caulobacter crescentus

Summary A recombination-deficient (Rec^-) strain of Caulobacter crescentus has been isolated from a collection of mutants sensitive to ultraviolet irradiation. The Rec^- mutant fails to give recombinants following Cr30-mediated generalized transduction or following RP4-mediated conjugation. The recombination frequency in the Rec^- strain is at least 5000-fold lower than in the wild type strains. The Rec^- mutant is indistinguishable from wild type in terms of morphology, growth rate, viability, and phage sensitivities, differing only in properties known to be associated with recA-type mutations in other organisms: recombination frequency, ultraviolet sensitivity, and Weigle reactivation. The map location of the rec-526 allele has not been identified, but rec-526 can be cotransferred with the fla-169 mutation by RP4-mediated conjugation at low frequency. This apparent linkage has been used to move the rec mutation to other strains. The Rec^- mutant resembles recA strains of other organisms and provides a healthy strain severely deficient in recombination for use in complementation and cloning studies involving C. crescentus.     

DNA degradation in minicells of Escherichia coli K-12

Summary The properties of minicell producing mutants of Escherichia coli deficient in gentic recombination were examined. Experiments were designed to test recombinant formation in conjugal crosses, survival following UV-irradiation in cells, and the state of DNA metabolism in minicells. The REC^- phenotypes are unaffected by min ⁺/^- genotypes in whole cells. In contrast to minicells produced by rec ⁺ parental cells, minicells from a recB21 strain have limited capacity to degrade linear, Hfr transferred DNA. The lack of a functional recA gene product, presumably involved in inhibiting the recBC nuclease action(s), permits unrestricted Hfr DNA breakdown in minicells produced by a recA1 strain. This results in an increase in TCA soluble products and in the formation of small DNA molecules that sediment near the top of an alkaline sucrose gradient. Unlike the linear DNA, circular duplex DNA from plasmids R64-11 or dv, segregated into the minicells, is resistant to breakdown. By using in vitro criteria, and [³²P]-labelled linear DNA from bacteriophage T₇ for substrate, we found that the ATP-dependent exonuclease of the recBC complex (exo V) is present in rec ⁺ and recA^- minicells, and is lacking in the recB21 mutant. In fact, the absence of a functional exo V in recBC^- minicells results in isolation of larger than average Hfr DNA from minicells. We suggest that recombination (REC) enzymes segregate into the polar minicells at the time of minicell biogenesis. This system should be useful for studies on DNA metabolism and functions of the recBC and recA gene products.Paper 1 in series, see Khachatourians et al., 1974.     

Isolation and characterization of Hfr males in Citrobacter freundii

Citrobacter freundii Hfr donor strains were isolated from a C. freundii strain harbouring a temperature-sensitive factor F ts 114 lac ⁺, by selecting for integrative suppression of the ts 114 mutation. Three Hfr strains were characterized, which transfer their chromosomes in a linear and oriented order. The first strain transfers: O-aro ⁺-ilv ⁺-pur ⁺-thr ⁺-leu ⁺-pro ⁺, the second: O-ilv ⁺-pur ⁺-thr ⁺-leu ⁺-pro ⁺ and the third: O-ilv ⁺-aro ⁺-nad ⁺-his ⁺-pro ⁺. The whole chromosome is transferred into the recipient cell within about 145 minutes. From these results we concluded that the linkage map of C. freundii is circular. Mating-pair formation on a membrane filter resulted in more recombinants being formed as compared with mating-pair formation in liquid medium. Furthermore the mating-pairs formed on a membrane were more stable. From one Hfr strain heterogenic F-prime factors could be isolated bearing the F ts 114 lac ⁺ genes from Escherichia coli and the pur ⁺ and/or ilv ⁺ genes from C. freundii. Preliminary mapping by interrupted mating indicated that the linkage map of C. freundii is in general very similar to those of E. coli, Salmonella typhimurium and Klebsiella aerogenes.     

Effect of L-ethionine the expression of the SOS system in Escherichia coli

The effect of L-ethionine, the ethyl analog of the essential amino acid methionine the SOS system of Escherichia coli was studied. This compound does not induce either inhibition of cell division nor cessation of cell respiration in a RecA⁺ Met⁺ RelA⁺ strain, nor in RecA⁺ Met⁻ RelA⁺ or RecA⁺ Met⁻ RelA⁻ mutants. Nevertheless, L-ethionine blocks the expression of both cited SOS functions in a recA441 mutant when it is growing at the restrictive temperature of 42°C. Furthermore, the inhibitory effect of the L-ethionine on the induction of the SOS system in this mutant is increased when the cells are preincubated for several hours in the presence of the analog, before the temperature shift. Moreover, cultures of the recA441 mutant incubated at 42°C in the presence of both L-ethionine and L-methionine present the same behaviour as the cultures of this mutant growing at the same temperature but without either amino acid. On the other hand, L-ethionine does not have any effect on the expression of the two mentioned SOS functions when these are induced by UV-irradiation in a RecA⁺ strain even if this compound is added to the cells several hours before irradiation.     

Tetracycline-resistance inEscherichia coli; a genetic contribution

A non-transmissible tetracycline-resistance plasmid inE. coli was found to be transmissible by transduction and by conjugation with the aid of theE. coli K12 sex-factor. Transfer of the tetracycline-resistance plasmid (R-tet) by transduction or conjugation to anE. coli K12 Hfr strain revealed that the plasmid was incompatible with the integrated F-factor. Selection for tetracycline-resistance after conjugation or transduction yielded Hfr colonies which carried the tetracycline-resistance determinant as a chromosomal marker. The tetracycline-resistance determinant was integrated at the 1 min region of theE. coli chromosome map (Taylor and Trotter, 1967) between the markersara andleu. Apart from Hfr colonies with a chromosomal tetracycline-resistance determinant, F-gal⁺-mediated transfer of R-tet to strain Hfr R4 gave some colonies in which the tetracycline-resistance determinant was carried on a fused plasmid that, besides the resistance determinant, contained thegal ⁺ marker of the original F-gal ⁺. This fused plasmid is transmissible and confers to an F⁻ cell male-specific phage-sensitivity, like an F-factor does. It is suggested that this fused plasmid, which is compatible with the integrated F-factor in the Hfr R4 cells, arose by recombination between F-gal ⁺ and R-tet.     

Some Properties of Excision-defective Recombination-deficient Mutants of Escherichia coli K-12

Strains of Escherichia coli that carry the mutation uvrA6 show no measurable excision of pyrimidine dimers and are easily killed by ultraviolet (UV) light, whereas strains that carry recA13 are defective in genetic recombination and are also UV-sensitive. An Hfr strain carrying uvrA6 was crossed with an F⁻ strain carrying recA13. Among the recombinants identified, one carrying uvrA recA proved to be of exceptional sensitivity to UV light. It is estimated from the UV dose (0.2 erg/mm² at 253.7 nm) required to reduce the number of colony-forming cells by one natural logarithm that about 1.3 pyrimidine dimers were formed in a genome of 5 × 10⁶ base pairs for each lethal event. This double mutant is 40 times more UV-sensitive than the excision-defective strain carrying uvrA6. The replication of one pyrimidine dimer is generally a lethal event in strains carrying recA13. Spontaneous breakdown and UV-induced breakdown of the deoxyribonucleic acid (DNA) of cells of the various genotypes were estimated by growing the cells in medium containing ³H-thymidine and measuring both acid-precipitable and acid-soluble radioactivity. The UV-induced degradation in strains with recA13 did not require the uvr⁺ genes and hence appears to depend upon a mechanism other than dimer excision. The greater level of survival after irradiation in Rec⁺ as compared to Rec⁻ bacteria may be due to a recovery mechanism involving the reconstruction of the bacterial chromosome through genetic exchanges which occur between the newly replicated sister duplexes and which effectively circumvent the damaged bases remaining in the DNA.     

Long-Term Field Release of Bioluminescent Sinorhizobium meliloti Strains to Assess the Influence of a recA Mutation on the Strains' Survival

A field release experiment was carried out to study the fate of the isogenic, firefly luciferase (luc) gene-tagged Sinorhizobium meliloti strains L1 (RecA⁻) and L33 (RecA⁺) in the environment. Both strains were released at concentrations of approximately 10⁶ cfu g⁻¹ soil in replicate and randomized field plots, which had been sown with alfalfa (Medicago sativa). The survival of both strains during the following 7 years could be subdivided into three phases: a sharp decline for more than two orders of magnitude within the first 4 months (phase I), followed by fluctuations around an average number of 10⁴ cfu g⁻¹ soil for nearly 4 years (phase II), and a further decline to approximately 60 cfu g⁻¹ (phase III). At most sampling dates, no significant differences in the survival of both strains were detected, indicating that the recA gene function was dispensable under these environmental conditions. During the field inoculation, both strains were dispersed accidentally by wind in small numbers to noninoculated field plots. Strain L33 established at a concentration of more than 10³ cfu g⁻¹ soil with subsequent seasonal fluctuations. Although strain L1 must have been disseminated to a similar extent, it could never be recovered from noninoculated field plots, indicating that the recA mutation interfered with the strain's capability to establish there. At the beginning of the field experiment, an indigenous alfalfa-nodulating population was below the limit of detection. In the following years, however, an indigenous population arose, which finally outcompeted both strains for saprophytic growth and alfalfa nodulation. RecA⁻ strain L1 was outcompeted for alfalfa nodulation slightly faster than its RecA⁺ counterpart L33. The diversity of the indigenous population was characterized by employing the Enterobacterial Repetitive Intergenic Consensus polymerase chain reaction fingerprint method. Typing of 2731 root nodule isolates revealed a total of 38 fingerprint groups. More than 80% of the isolates could be grouped into six dominant fingerprint groups, indicating that a few dominant bacterial strain types had outcompeted the released strains.     

Role of surface exclusion genes in lethal zygosis in Escherichia coli K12 mating

Summary We find that diaminopimelic acid in the recipient membrane is released into the medium during bacterial matings, indicating that membrane damage was inflicted on the recipient by the donor, probably for forming a channel for DNA transfer. When the damage is extensive, as in matings with an excess of Hfr bacteria, the F^- bacteria are killed (lethal zygosis). The transfer of a large amount of DNA in Hfr matings appears to enhance the killing. In analogous F⁺xF^- (Nal^r) matings, on the other hand, killing of F^- bacteria does not occur unless F plasmid transfer is inhibited by a substance like nalidixic acid. The F^- bacteria are killed, suggesting that F plasmids contain genes that express immunity to lethal zygosis in the recipient. For example, bacteria containing surface exclusion-deficient mutants of F plasmids, such as traS ^- and traT ^-, induce lethal zygosis in F^- bacteria and are susceptible to it. Various tra ^- polar mutants that abolish surface exclusion are also susceptible to lethal zygosis when mated with Hfr bacteria. Kinetic experiments indicate that in F⁺ (wild type) x F^- matings, immunity to lethal zygosis is expressed in the F^- recipient within 1/4 division time, whereas a complete expression of surface exclusion requires more than 1 division time. Thus, a complete change in all receptor sites seems to be required for the expression of surface exclusion.     

Expression and regulation of the Escherichia coli glutamate dehydrogenase gene (gdh) in Rhizobium japonicum

The glutamate dehydrogenase (gdh) gene of Escherichia coli was transferred into an ammonium assimilation deficient mutant (Asm^-) of Rhizobium japonicum (CJ9) using plasmid pRP301, a broad host range derivative of RP₄. Exconjugants capable of growth on ammonia as sole N-source occurred at a frequency of 6.8×10^-6. Assimilatory GDH (NADP⁺) activity was detected in the strain carrying the E. coli gdh gene and the pattern of ammonia assimilation via GDH was similar to that of the Asm⁺ wild type strain. However, GDH mediated ammonia assimilation was not subject to regulation by l-glutamate. Nitrogenase activity was expressed ex planta in R. japonicum CJ9 harbouring the gdh gene, however, the presence of the gdh gene did not restore symbiotic effectiveness to the CJ9 Asm^- strain in nodules. The gdh plasmid was maintained in approximately 90% of the isolates recovered from soybean nodules.Abbreviations gdh glutamate dehydrogenase - Asm^- mutant ammonia assimilation deficient mutant