Utilization of a thermosensitive episome bearing transposon TN10 to isolate Hfr donor strains of Erwinia carotovora subsp. chrysanthemi

A thermosensitive episome bearing the transposon Tn10, F(Ts)::Tn10 Lac+, has been successfully transferred from Escherichia coli to several wild strains of the enterobacteria Erwinia carotovora subsp. chrysanthemi, which are pathogenic on Saintpaulia ionantha. In one of these strains, all of the characters controlled by this episome (Lac+, Tetr, Tra+) were expressed, and its replication was stopped at 40 degrees C and above. At 30 degrees C, the episome was easily transferred between strains derived from E. carotovora subsp. chrysanthemi 3937j and to E coli. Hfr donor strains were obtained from a F' strain of 3937j by selecting clones which grew at 40 degrees C on plates containing tetracycline. One of these strains, Hfrq, was examined in more detail: the characters Lac+ and Tetr were stabilized and did not segregate higher than its parental F' strain. The mating was most efficient at 37 degrees C on a membrane. Hfrq transferred its chromosome to recipient strains at high frequency and in a polarized fashion, as evidenced by the gradient of transfer frequencies, the kinetics of marker entry (in interrupted mating experiments), and the analysis of linkage between different markers. The chromosome of Hfrq was most probably transferred in the following sequence: origin...met...xyl...arg...ile...leu...thr...cys...pan...ura...gal...trp...his. ..pur... Moreover, this genetic transfer system proved to be efficient in strain construction.     

Recombinant DNA transfer to Escherichia coli of human faecal origin in vitro and in digestive tract of gnotobiotic mice

Abstract The role of helper elements in the mobilisation of pBR recombinant plasmids ( tra ⁻, mob ⁻, ori T⁺ and tra ⁻, mob ⁻, ori T⁻) from genetically engineered Escherichia coli K12 strains to other K12-strains and to wild-type E. coli strains of human faecal origin was examined. Transfer experiments were done in the digestive tract of axenic (germ free) and gnotobiotic mice, associated with human faecal flora, HFF. The kinetics of implantation of donors, recipients and transconjugants were determined. Mobilisation of ori T⁺ pBR-type plasmids, by trans-complementation with the products of tra and mob genes was obtained with E. coli K12, in the digestive tract of axenic mice and the resulting transconjugants became established together with the recipient and donor strains. Such mobilisation was only observed sporadically with one E. coli of human origin in axenic mice, but did not occur in gnotobiotic HFF mice. The E. coli strains of human origin were able to promote transfer of an ori T⁻ pBR-type plasmid in vitro but not in axenic or gnotobiotic mice. Transconjugants of wild-type strains obtained in in vitro mating experiments and inoculated into gnotobiotic HFF mice were eliminated as rapidly as the recombinant K12 strains. This work indicates that ≥ 50% of wild-type E. coli strains were able to promote transfer of pBR ori T⁻ plasmids in vitro.     

Description of an incompatibility mutant of Escherichia coli

A mutant Hfr strain of Escherichia coli which has an impaired incompatibility function but is normal for other F factor functions has been isolated. This Inc(-) Hfr permits the maintenance and transfer of both the integrated F factor and an F' factor. F' factors have been isolated from the integrated F factor of the Inc(-) Hfr strain. When these episomes were tested in matings with Hfr or F' strains, they did not differ in any observed way from wild-type F' factors.     

Evidence for the Presence of Nontranslated T7 Late mRNA in Infected F′(PIF+) Episome-Containing Cells

The inability of T7 to develop in cells of Escherichia coli containing F(+) or substituted F' episomes is a result of the failure to synthesize late proteins; no in vivo translation of mRNA species synthesized by the T7 RNA polymerase occurs. Further experiments have been performed to measure the amount of late mRNA in T7-infected F'(PIF(+)) cells. (We have designated the property of phage inhibition of F factors as PIF; the wild-type episome is therefore F'[PIF(+)].) T7 late proteins were synthesized in vitro by using a system programed with RNA extracted from T7-infected F(-) and F'(PIF(+)) cells. The T7 lysozyme, product of gene 3.5, and the gene 10 head protein were assayed. The following results were obtained: (i) mRNA capable of supporting in vitro synthesis of lysozyme and the gene 10 head protein is present in T7-infected F'(PIF(+)) cells; (ii) lysozyme mRNA extracted from T7-infected F'(PIF(+)) cells is present at 70 to 75% of the level found in T7-infected F(-) cells; (iii) gene 10 mRNA is present at 35 to 78% of the level found in T7-infected F(-) cells. No in vivo synthesis of either lysozyme or gene 10 protein can be detected in T7-infected F'(PIF(+)) cells although normal synthesis of these proteins occurs in F(-) cells. These findings confirm that the block in T7 development in F'(PIF(+)) cells results from the failure to translate late classes of T7 RNA.     

PROPERTIES OF F' STRAINS OF ESCHERICHIA COLI SUPERINFECTED WITH F-LACTOSE AND F-GALATOSE EPISOMES

Echols, Harrison (University of Wisconsin, Madison). Properties of F' strains of Escherichia coli superinfected with F-lactose and F-galactose episomes. J. Bacteriol. 85:262-268. 1963.-A study of F' superinfection of F' strains of Escherichia coli by F Lac P and F Gal indicates that superinfecting F Lac P may achieve a stable existence along with native F Lac P in approximately 1% of the recipient F' population. The stable presence of F Gal, on the other hand, leads to a displacement or suppression of F Lac P when F Gal is used as a superinfecting agent or as the native episome. The majority of F' bacteria used as recipients neither acquire in stable form the superinfecting F-linked genes nor demonstrate gene activity immediately after attempted transfer, as judged by alkaline phosphatase synthesis directed by an F-transferred P(+) gene. This failure to show gene activity suggests that the F' bacteria which are sterile as recipients exclude transfer rather than inhibit subsequent multiplication.     

胸膜肺炎放线杆菌萘啶酸抗性菌株的选育和信号标签突变株的构建

胸膜肺炎放线杆菌(APP)是重要的猪呼吸道病原菌,给世界养猪业造成严重的经济损失.信号标签突变(STM)技术是在宿主动物体内鉴定病原菌毒力因子的高通量方法.通过体外传代选育出APP血清1型和3型萘啶酸抗性菌株,再以萘啶酸抗性菌株为受体菌,以携带mini-Tn10的标签质粒(pLOF/TAG1-48)的E.coli CC118 λ pir或S17-1λpir为供体菌,在或不在E.coli DH5α(pRK2073)的辅助下,进行三亲本或两亲本接合,通过抗性筛选、PCR和Southern杂交鉴定转座突变株.结果表明:体外萘啶酸加压传代很容易选育出萘啶酸抗性APP菌株,该抗性的产生与DNA促旋酶A亚基基因gyrA的突变有关.在APP与E. coli接合实验中,两亲本接合比三亲本接合操作更简单,效率也较高;APP不同菌株在接合和转座效率上存在很大差异,血清1型菌株高于血清3型菌株,3型标准菌株高于地方分离株JL03-R.本研究为APP STM突变体库的构建与毒力基因的鉴定奠定了基础.     

In vitro and in situ conjugal transfer of an R-plasmid among enterobacteriaceae species isolated from meat samples

Abstract An R-plasmid donor strain of Escherichia coli isolated from a meat sample was mated with potential bacterial recipients belonging to the family Enterobacteriaceae isolated from ground beef and chicken samples. Nine different strains having different plasmid profiles were used as recipients in broth conjugation experiments. The recipients were identified as Enterobacter cloacae, Hafnia alvei, E. coli, Klebsiella pneumoniae and K. oxytoca . Of 1250 ampicillin resistant, tetracycline sensitive colonies tested, the incidence of recipients was estimated to be 3% (in ground beef) and 11% (in chicken) of the bacteria population. Two of the recipients, E. coli and K. Oxytoca also behaved as donors and transferred their R-plasmids to a laboratory recipient strain of E. coli K12-711. In vitro R-plasmid transfer frequencies varied within a wide range, from 10⁻² to 10⁻⁷ among recipients. Generally, frequencies of plasmid transfer were highest at 30°C and declined with decreasing temperature. Three of the recipient isolates, E. cloacae, H. alvei and E. coli displayed transfer of R-plasmids at 10°C in broth matings. Similar trends in R-plasmid transfer frequencies also were observed under in situ mating conditions in raw ground beef and pasteurized milk samples.     

Influence of salts and temperature on the transfer of mercury resistance from a marine pseudomonad to Escherichia coli

Thirty-one strains of marine bacteria were examined for their ability to transfer mercury resistance to Escherichia coli in complex media; eight strains were able to transfer their resistance marker, with frequencies ranging from 10(-3) to 10(-8). Frequencies generally increased with the increase of the mating period. Additional mating experiments were carried out with one strain, belonging to the pseudomonads, to estimate the influence of temperature, salinity, and time on the conjugal transfer frequency of mercury resistance markers. The higher frequencies occurred at 30 degrees C, in a salt medium (37%), after 24 h of mating.     

Influence of salts and temperature on the transfer of mercury resistance from a marine pseudomonad to Escherichia coli.

Thirty-one strains of marine bacteria were examined for their ability to transfer mercury resistance to Escherichia coli in complex media; eight strains were able to transfer their resistance marker, with frequencies ranging from 10(-3) to 10(-8). Frequencies generally increased with the increase of the mating period. Additional mating experiments were carried out with one strain, belonging to the pseudomonads, to estimate the influence of temperature, salinity, and time on the conjugal transfer frequency of mercury resistance markers. The higher frequencies occurred at 30 degrees C, in a salt medium (37%), after 24 h of mating.     

Propagation of ribonucleic acid coliphages in gnotobiotic mice.

To clarify the propagation cycle of bacteriophages in their natural habitats, we tested whether animals could support ribonucleic acid (RNA) phage propagation in their intestines, using germfree mice as the test animal. Propagation of four different antigenic types of RNA phages was tested. No detectable propagation or colonization of RNA phages was observed either in germfree mice or in gnotobiotic mice infected with the F- strain of Escherichia coli. Propagation or colonization was observed when RNA phages were orally introduced into gnotobiotic mice harboring the F+ or F' strain of E. coli. These results were consistent with data for in vitro propagation experiments. Fecal titers of phages were monitored over 24 to 98 days and were found to vary from 10(5) to 10(11) plaque-forming units per g of feces. Streptomycin administration gradually led to the disappearance of bacteria and, concomitantly, the RNA phages. Phages recovered from gnotobiotic mice feces included some of novel antigenic types. The bacterial isolates recovered from gnotobiotic mice harboring F+ bacteria included the original F+ strain, strains which had become F-, and some which had become inefficient hosts for the propagation of RNA phages.     

Propagation of ribonucleic acid coliphages in gnotobiotic mice.

To clarify the propagation cycle of bacteriophages in their natural habitats, we tested whether animals could support ribonucleic acid (RNA) phage propagation in their intestines, using germfree mice as the test animal. Propagation of four different antigenic types of RNA phages was tested. No detectable propagation or colonization of RNA phages was observed either in germfree mice or in gnotobiotic mice infected with the F- strain of Escherichia coli. Propagation or colonization was observed when RNA phages were orally introduced into gnotobiotic mice harboring the F+ or F' strain of E. coli. These results were consistent with data for in vitro propagation experiments. Fecal titers of phages were monitored over 24 to 98 days and were found to vary from 10(5) to 10(11) plaque-forming units per g of feces. Streptomycin administration gradually led to the disappearance of bacteria and, concomitantly, the RNA phages. Phages recovered from gnotobiotic mice feces included some of novel antigenic types. The bacterial isolates recovered from gnotobiotic mice harboring F+ bacteria included the original F+ strain, strains which had become F-, and some which had become inefficient hosts for the propagation of RNA phages.     

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.     

Elimination of Sex Factors in Escherichia coli by Urea

Eliminatory action of urea on the sex factor (F) in Escherichia coli K-12 strains is reported. Growth of E. coli harboring F or F'8 (F-gal) factors in Penassay Broth containing urea led to the loss of these genetic elements and yielded F(-) cells. Appearance of F(-) cells among survivors was already observed when the culture was in the very early stage of exponential phase. However, frequencies of F(-) cells formed did not increase much as a function of the incubation time. Unusual F(+) or F'8 cells which retained the ability of genetic transfer but showed resistance to M12 phage were also isolated. Addition of sucrose to broth with urea led to the favorable growth of cells in the culture and the increase, if little, of elimination frequencies of F factors by urea. These findings, coupled with other observations, suggest that urea has two separate actions in enhancing the frequency of F(-) bacteria, namely, (i) to inactivate F by direct action, such as mutation, and (ii) to select the F(-) variants by differentially inhibiting the growth of F(+).     

Complementation of nitrogen-regulatory (ntr-like) mutations in Rhodobacter capsulatus by an Escherichia coli gene: cloning and sequencing of the gene and characterization of the gene product.

In vivo genetic engineering by R' plasmid formation was used to isolate an Escherichia coli gene that restored the Ntr+ phenotype to Ntr- mutants of the photosynthetic bacterium Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata; J. F. Imhoff, H. G. Trüper, and N. Pfenning, Int. J. Syst. Bacteriol. 34:340-343, 1984). Nucleotide sequencing of the gene revealed no homology to the ntr genes of Klebsiella pneumoniae. Furthermore, hybridization experiments between the cloned gene and different F' plasmids indicated that the gene is located between 34 and 39 min on the E. coli genetic map and is therefore unlinked to the known ntr genes. The molecular weight of the gene product, deduced from the nucleotide sequence, was 30,563. After the gene was cloned in an expression vector, the gene product was purified. It was shown to have a pI of 5.8 and to behave as a dimer during gel filtration and on sucrose density gradients. Antibodies raised against the purified protein revealed the presence of this protein in R. capsulatus strains containing the E. coli gene, but not in other strains. Moreover, elimination of the plasmid carrying the E. coli gene from complemented strains resulted in the loss of the Ntr+ phenotype. Complementation of the R. capsulatus mutations by the E. coli gene therefore occurs in trans and results from the synthesis of a functional gene product.     

The production of F41 fimbriae by piglet strains of enterotoxigenic Escherichia coli that lack K88, K99 and 987P fimbriae

The enterotoxigenic Escherichia coli strains 1676, 1706, 1751 and KEC96a, which do not produce fimbrial adhesive antigens of the K88, K99 or 987P antigen type reacted both in vitro and in vivo with antiserum to F41 fimbriae in an indirect immunofluorescent antibody technique. Antiserum used to demonstrate material B, an adhesive antigen thought to mediate the adhesive and mannose-resistant (MR) haemagglutinating properties of E. coli strains 1676, 1706 and 1751, reacted in vitro with an F41+ strain. The antiserum also inhibited the MR haemagglutinating activity of F41 antigen and gave an anionic precipitation line in immunoelectrophoresis experiments with an extract containing F41 antigen. The MR haemagglutinating properties of an antigen extract containing material B from E. coli strain 1706 was neutralized by antiserum to F41 fimbriae and by OK antisera to E. coli strains that produce both F41 and K99 fimbriae. These sera also gave an anionic precipitation line with the MR haemagglutinin from E. coli strain 1706 and the MR haemagglutinin gave a line of identity with F41 in gel diffusion experiments with antiserum to F41 fimbriae. OK antisera to K99+ F41- bacteria and OK antisera to K88+ bacteria and 987P+ bacteria did not react with this haemagglutinin. Transmission electron microscopy on the ileum of newborn gnotobiotic piglets infected with E. coli strain 1706 showed irregular, poorly defined filamentous material surrounding some,though not all, bacteria but regular fimbrial structures were not visible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and characterization of the gene coding for the aerobic azoreductase from Xenophilus azovorans KF46F

The gene coding for an aerobic azoreductase was cloned from Xenophilus azovorans KF46F (formerly Pseudomonas sp. strain KF46F), which was previously shown to grow with the carboxylated azo compound 1-(4'-carboxyphenylazo)-2-naphthol (carboxy-Orange II) as the sole source of carbon and energy. The deduced amino acid sequence encoded a protein with a molecular weight of 30,278 and showed no significant homology to amino acid sequences currently deposited at the relevant data bases. A presumed NAD(P)H-binding site was identified in the amino-terminal region of the azoreductase. The enzyme was heterologously expressed in Escherichia coli and the azoreductase activities of resting cells and cell extracts were compared. The results suggested that whole cells of the recombinant E. coli strains were unable to take up sulfonated azo dyes and therefore did not show in vivo azoreductase activity. The turnover of several industrially relevant azo dyes by cell extracts from the recombinant E. coli strain was demonstrated.     

The function of cytoplasmic flavin reductases in the reduction of azo dyes by bacteria

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an "azo reductase." The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic "azo reductases," which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.     

Horizontal transfer of nonconjugative plasmids in a colony biofilm of Escherichia coli

We tested the possibility of nonconjugative lateral DNA transfer in a colony biofilm of mixed Escherichia coli strains. By simply coculturing a plasmid-free F(-) strain and another F(-) strain harboring a nonconjugative plasmid in a colony biofilm on antibiotic-free agar media, transformed cells were produced within 24-48 h at the frequency of 10(-10)-10(-9) per recipient cell. PCR analysis of the transformed cells demonstrated the occurrence of lateral plasmid transfer. These cells survived until at least day 7 under antibiotic-free conditions. Liquid cultures of the same strains in Luria-Bertani broth produced no or few transformants, suggesting the importance of colony-biofilm formation for plasmid transfer. This is a novel line of evidence indicating that nonconjugative, nonviral horizontal gene transfer can occur between E. coli cells.     

Recombination and the Escherichia coli K-12 sex factor F.

Recombination between two Flac tra minus elements to give Flac tra plus recombinants was measured in Rec plus and Rec minus strains of Escherichia coli K-12. Polar tra mutations were used to increase the proportion of tra plus recombinants among the parental Flac tra minus elements transferred by complementation. The kinetics, measured in a rec plus strain, showed that recombination began about 1 h after the initiation of mating and was completed about 1 h later. Recombination was abolished in a recA minus strain, reduced by two-thirds in a recF minus strain, and unaffected in recB minus and recC minus strains. It is proposed that the part not due to the RecF pathway results from a RecBC- and RecF-independent system for formation of single-stranded joins. One such join could be followed either by transfer and a site-specific recombination event, or by a second single-stranded join and then transfer: in either case replication and inheritance of the recombinant molecule would be dependent upon the F transfer replication system. Chromosome mobilization by an F' element was normal in a recB plus recF minus strain, and was reduced only fourfold in a recB minus recF plus strain: in the latter strain, both the RecF pathway and the system for single-stranded joins may have contributed to mobilization. Measurement of post-conjugational chromosomal recombination in exponential-phase recipient cells carrying surface exclusion-deficient Flac mutants indicated that F does not itself determine a generalized recombination system able to replace the RecA plus product or the RecBC and RecF pathways.