Stepwise selection of defective nitrogen-fixing phenotypes in Escherichia coli K-12 by dimethyl sulfoxide.

Storage in dimethyl sulfoxide of Escherichia coli K-12 hybrids carrying Klebsiella pneumoniae nif+ genes can result in selection of a defective nitrogen-fixing phenotype. Dimethyl sulfoxide appears to affect particular inner membrane proteins associated with energy metabolism in E. coli K-12.     

Genetic determinants of the synthesis of the polysaccharide capsular antigen K27(A) of Escherichia coli.

Most of the his+ hybrids from crosses between the Escherichia coli donor Hfr45(O8:K27) and different E. coli O9 recipients expressed the donor O8 antigen specificity and produced the capsular antigen K27. Therefore these hybrids must have inherited the his-linked donor rfb region determining the synthesis of O8- specific polysaccharides as well as his-linked genes involved in K27 antigen synthesis. In the living state these hybrids were inagglutinable in O8 antiserum like the donor cells. However, when E. coli K12 and O8:K42- were used as recipients most of the his+ hybrids were agglutinable in O8 and K27 antisera. The amounts of K27 antigen present in these hybrids, designated as K27i (intermediate) forms, were sufficient to evoke the production of K27 antibodies in rabbits, but insufficient to inhibit O-agglutination of the respective cells. The additional transfer of the trp region of E. coli O8:K27 into such K27i forms frequently resulted in O-inagglutinable K27+ hybrids. This is attributed to the introduction of trp-linked genes which apparently play a role in the synthesis of K27 capsular antigen. Tus it is concluded that at least two gene loci, one close to his and the other close to trp, are required for the synthesis of the complete capsular antigen K27.     

Pathways of energy metabolism required for phenotypic expression of nif+Kp genes in Escherichia coli.

In E. coli K12 (F'nif+Kp) hybrids, electron-transport-dependent phosphorylation is not necessary for anaerobic nitrogen fixation, and substrate level phosphorylation can provide sufficient ATP from glucose for nitrogenase activity. The fumarate-reduction system, however, is essential in these hybrids for the transfer of electrons to nitrogenase. This system is probably also involved in maintaining the membrane in the energized state, thereby allowing nitrogen fixation to occur. The nitrate-reduction system, which can energize the membrane like the fumarate-reduction system, is not necessary for nitrogenase activity in the E. coli K12(F'nif+Kp) hybrids. However, two nitrate reductase genes, chlA, and chlB, are essential for inhibition of nitrogen fixation by nitrate. Moreover, nitrate inhibits nitrogenase activity and this inhibition is most probably effected through a regulator factor coded by chlA and chlB.     

A gene fusion approach to the study of pullulanase export and secretion in Eschenchia coli

A series of fusions between the gene for the Klebsiella pneumoniae secreted lipoprotein pullulanase (pulA) and the genes for cytoplasmic beta-galactosidase (lacZ) or periplasmic alkaline phosphatase (phoA) were created by transposon mutagenesis using mini-MudII1681 or TnphoA, respectively. The hybrid genes were expressed in Escherichia coli K-12 with or without the K. pneumoniae genes that promote pullulanase secretion in E. coli. We characterized seven different pulA-lacZ gene fusions encoding hybrid polypeptides containing from 14 to c. 1060 residues of pro-pullulanase. All but the smallest hybrid were fatty acylated and were toxic to producing cells, causing the accumulation of precursors of other exported proteins. Four different pulA-phoA gene fusions encoded hybrids with alkaline phosphatase activity. All four hybrids were fatty acylated, but were not toxic. Although the hybrids were apparently membrane-associated, they were not secreted into the medium either by E. coli carrying pullulanase secretion genes or by K. pneumoniae. Immunofluorescence tests indicated that the pullulanase secretion genes promoted the localization of one of these hybrids to the outer face of the E. coli outer membrane, which may have important implications for the design of live vaccine strains and of immobilized enzymes.     

Genetic Transfer of Salmonella typhimurium and Escherichia coli Lipopolysaccharide Antigens to Escherichia coli K-12

Escherichia coli K-12 varkappa971 was crossed with a smooth Salmonella typhimurium donor, HfrK6, which transfers early the ilv-linked rfa region determining lipopolysaccharide (LPS) core structure. Two ilv(+) hybrids differing in their response to the LPS-specific phages FO and C21 were then crossed with S. typhimurium HfrK9, which transfers early the rfb gene cluster determining O repeat unit structure. Most recombinants selected for his(+) (near rfb) were agglutinated by Salmonella factor 4 antiserum. Transfer of an F' factor (FS400) carrying the rfb-his region of S. typhimurium to the same two ilv(+) hybrids gave similar results. LPS extracted from two ilv(+),his(+), factor 4-positive hybrids contained abequose, the immunodominant sugar for factor 4 specificity. By contrast, his(+) hybrids obtained from varkappa971 itself by similar HfrK9 and F'FS400 crosses were not agglutinated by factor 4 antiserum, indicating that the parental E. coli varkappa971 does not have the capacity to attach Salmonella O repeat units to its LPS core. It is concluded that the Salmonella rfb genes are expressed only in E. coli varkappa971 hybrids which have also acquired ilv-linked genes (presumably rfa genes affecting core structure or O-translocase ability, or both) from a S. typhimurium donor. When E. coli varkappa971 was crossed with a smooth E. coli donor, Hfr59, of serotype O8, which transfers his early, most his(+) recombinants were agglutinated by E. coli O8 antiserum and lysed by the O8-specific phage, Omega8. This suggests that, although the parental E. coli K-12 strain varkappa971 cannot attach Salmonella-specific repeat units to its LPS core, it does have the capacity to attach E. coli O8-specific repeat units.     

Behavior of coliphage lambda in hybrids between Escherichia coli and Salmonella

Salmonella typhosa hybrids able to adsorb lambda were obtained by mating S. typhosa recipients with Escherichia coli K-12 donors. After adsorption of wild-type lambda to these S. typhosa hybrids, no plaques or infective centers could be detected. E. coli K-12 gal(+) genes carried by the defective phage lambdadg were transduced to S. typhosa hybrids with HFT lysates derived from E. coli heterogenotes. The lysogenic state which resulted in the S. typhosa hybrids after gal(+) transduction differed from that of E. coli. Ability to produce lambda, initially present, was permanently segregated by transductants of the S. typhosa hybrid. S. typhosa lysogens did not lyse upon treatment for phage induction with mitomycin C, ultraviolet light, or heat in the case of thermoinducible lambda. A further difference in the behavior of lambda in Salmonella hybrids was the absence of zygotic induction of the prophage when transferred from E. coli K-12 donors to S. typhosa. A new lambda mutant class, capable of forming plaques on S. typhosa hybrids refractory to wild-type lambda, was isolated at low frequency by plating lambda on S. typhosa hybrid WR4254. Such mutants have been designated as lambdasx, and a mutant allele of lambdasx was located between the P and Q genes of the lambda chromosome. Plaques were formed also on the S. typhosa hybrid host with a series of lambda(i21) hybrid phages which contain the N gene of phage 21. The significance of these results in terms of Salmonella species as hosts for lambda is discussed.     

Genetic mapping of tyramine oxidase and arylsulfatase genes and their regulation in intergeneric hybrids of enteric bacteria.

The genes for arylsulfatase (atsA) and tyramine oxidase (tynA) have been mapped in Klebsiella aerogenes by P1 transduction. They are linked to gdhD and trp in the order atsA-tynA-gdhD-trp-pyrF. Complementation analysis using F' episomes from Escherichia coli suggested an analogous location of these genes in E. coli, although arylsulfatase activity was not detected in E. coli. P1 phage and F' episomes were used to create intergeneric hybrid strains of enteric bacteria by transfer of the ats and tyn genes between K. aerogenes, E. coli, and Salmonella typhimurium. Intergeneric transduction of the tynK gene from K. aerogenes to an E. coli restrictionless strain was one to two orders less frequent than that of the leuK gene. The tyramine oxidase of E. coli and S. typhimurium in regulatory activity resemble very closely the enzyme of K. aerogenes. The atsE gene from E. coli was expressed, and latent arylsulfatase protein was formed in K. aerogenes and S typhimurium. The results of tyramine oxidase and arylsulfatase synthesis in intergeneric hybrids of enteric bacteria suggest that the system for regulation of enzyme synthesis is conserved more than the structure or function of enzyme protein during evolution.     

Antibiotic resistant mutants of Escherichia coli K12 show increases in heterologous gene expression

Using a variety of antibiotics, it was found that nine separate isolates of spontaneous antibiotic resistant mutants of Escherichia coli K12 pPSX-vioABCDE overproduce the anti-tumour antibiotic violacein. Subsequent analysis showed that seven of these mutations occurred on the plasmid pPSX-vioABCDE. The other two overproducing strains carried spontaneous chromosomal mutations to lincomycin and kanamycin. The kanamycin resistant mutant of E. coli K12 DH10B (AA23) and a lincomycin resistant mutant of E. coli K12 LE392 (AA24) increased the synthesis of violacein. The plasmid pPSX-vioABCDE opv-1 contains a violacein over-production (opv-1) mutation which when introduced into either E. coli K12 AA23 or AA24, resulted in a hyper-production of violacein. Remarkably, E. coli K12 AA23 pPSX-vioABCDE opv-1 produced 41 times the normal level of violacein. In addition, both E. coli K12 AA23 and E. coli K12 AA24 demonstrated an increase in expression of an alpha amylase gene from Streptomyces lividans and the urease gene cluster from Klebsiella oxytoca. These results suggest that selection of antibiotic resistant mutants can increase heterologous gene expression in E. coli K12. Additionally, the increased expression is a general effect applicable to genes and gene clusters cloned into E. coli K12 from both Gram-positive and Gram-negative bacteria.     

A chimeric Anabaena/ Escherichia coli KdpD protein (Anacoli KdpD) functionally interacts with E. coli KdpE and activates kdp expression in E. coli

The kdpFABC operon, coding for a high-affinity K(+)-translocating P-type ATPase, is expressed in Escherichia coli as a backup system during K(+) starvation or an increase in medium osmolality. Expression of the operon is regulated by the membrane-bound sensor kinase KdpD and the cytosolic response regulator KdpE. From a nitrogen-fixing cyanobacterium, Anabaena sp. strain L-31, a kdpDgene was cloned (GenBank accession no. AF213466) which codes for a KdpD protein (365 amino acids) that lacks both the transmembrane segments and C-terminal transmitter domain and thus is shorter than E. coli KdpD. A chimeric kdpD gene was constructed and expressed in E. coli coding for a protein (Anacoli KdpD), in which the first 365 amino acids of E. coli KdpD were replaced by those from Anabaena KdpD. In everted membrane vesicles, this chimeric Anacoli KdpD protein exhibited activities, such as autophosphorylation, transphosphorylation and ATP-dependent dephosphorylation of E. coli KdpE, which closely resemble those of the E. coli wild-type KdpD. Cells of E. coli synthesizing Anacoli KdpD expressed kdpFABC in response to K(+) limitation and osmotic upshock. The data demonstrate that Anabaena KdpD can interact with the E. coliKdpD C-terminal domain resulting in a protein that is functional in vitro as well as in vivo.     

Genetic analysis of Escherichia coli O111:B4, a strain of medical and biochemical interest.

Procedures have been worked out which allow, for the first time, the genetic analysis of Escherichia coli O111:K58:H2 (O111:B4). The approximate map position of mutant loci was determined by mating with 15 Hfr strains of E. coli K-12. In addition, P1 transduction procedures were used for establishing relative gene order and linkage for any region of the E. coli O111:B4 chromosome. To obtain these, it was necessary to select for a rare P1 lysogen since E. coli O111:B4 is resistant to phage P1. Finally, genetic homology between E. coli strains K-12 and O111:B4 is suggested since they can form stable haploid hybrids, and several loci have similar map positions in the two strains.     

Isolation of Circular Deoxyribonucleic Acid from Salmonella typhosa Hybrids Obtained from Matings with Escherichia coli Hfr Donors

Heterozygous, partial diploid Salmonella typhosa hybrids obtained from matings with Escherichia coli K-12 Hfr strains were observed to contain supercoiled, circular deoxyribonucleic acid (DNA) when examined by the dye-buoyant density method. Examination of one such S. typhosa hybrid after its loss, by segregation, of the inherited E. coli genetic markers revealed a concurrent loss of its supercoiled circular DNA. Subsequent remating of this segregant with various E. coli Hfr strains resulted in the reappearance of the circular DNA. Molecular weight determinations of circular DNA molecules isolated from a number of S. typhosa partial diploid hybrids were made by sucrose density gradient ultracentrifugation and electron microscopy. These studies revealed a range of molecular sizes among the various hybrids examined, but each hybrid exhibited only a single characteristic size for its contained circular DNA. The range of size is consistent with the presence in each hybrid of a different length of E. coli chromosome. It was concluded that the E. coli Hfr genetic segments transferred to these S. typhosa hybrids were conserved, in the diploid state, in the form of supercoiled, circular DNA molecules.     

Pl-mediated Transduction of a Gene That Controls Radiation Sensitivity and Capsular Polysaccharide Synthesis from Shigella dysenteriae to Escherichia coli

When Shigella dysenteriae strain 60 is used as a donor and Escherichia coli K-12 strains that are ultraviolet (UV)-sensitive, mucoid, and proline-requiring (Pro(-)) are employed as recipients, selection for Pro(+) yields 2 to 6% nonmucoid clones. All of the nonmucoid clones examined are UV-resistant. Most of the nonmucoid UV-resistant transductants are partial diploids for the genes being studied. When these Shigella-Escherichia hybrids are used as donors with the same E. coli recipients, the cotransduction of Pro(+) and nonmucoidness is greatly increased (59 to 94% cotransduction). All of these nonmucoid transductants examined were also UV-resistant. The results indicate that Shigella contains an allele (designated ShproC(+)) homologous to proC of E. coli and a second linked allele (designated ShcapR(+)) homologous to the capR allele of E. coli. The ShcapR(+) allele changes the phenotype of certain E. coli strains from mucoid UV-sensitive (capR6) or very sensitive (capR9) to nonmucoid and UV-resistant. Unanticipated capR allele interactions in the partial diploid hybrids are described.     

Mobilization of phase I plasmid in Shigella sonnei and stability of its inheritance in rough strains of Shigella and Escherichia

The plasmid pSS120, determining the synthesis of species specific I phase antigen of Shigella sonnei is mobilized for genetic transfer into E. coli K12 recipient cells with the frequency 12-41%. The frequency depends on the type of mobilized plasmid and recipient strain. The I phase antigen is normally expressed in II phase recipient cells and in E. coli cells. During mobilization pSS120 forms cointegrates representing a recombinant of mobilizing and mobilized plasmids DNA. The study of pSS120 inheritance stability has shown the plasmid to be unstable during culturing of bacteria and to be partially lost from the parent Shigella sonnei strains as well as from the "hybrid" transconjugants obtained. The 60 Md plasmid present in the donor strains of Shigella sonnei is prone to structural fragmentation particularly expressed in Shigella sonnei/E. coli hybrids.     

Conservation and transfer of Escherichia coli genetic segments by partial diploid Hfr strains of Salmonella typhosa

Heterozygous, partial diploid hybrids were obtained in a Salmonella typhosa Hfr strain by using it as the recipient in a mating with the Escherichia coli Hfr donor WR2004 (O...proA...leu). Three of these S. typhosa Hfr hybrids were observed to mobilize and transfer the diploid E. coli genes, at high frequencies, to an E. coli recipient. The gradient of transfer frequencies of E. coli markers from these S. typhosa Hfr hybrids was similar to that observed with E. coli Hfr WR2004, from which they were derived. Interrupted matings with one of these S. typhosa Hfr hybrids, designated WR4272, showed the entry times for the proA, thr(-)leu, and argB E. coli diploid markers to be identical to the times obtained for these markers with E. coli Hfr WR2004. Also, the pattern of unselected inheritance of the diploid E. coli markers of S. typhosa Hfr hybrid WR4272 was similar to that observed with the chromosomal markers of E. coli Hfr WR2004. It was concluded that S. typhosa Hfr hybrid WR4272 contains, in addition to its Salmonella genome, a physically continuous E. coli chromosomal segment which is genetically complete from proA to at least the strA locus. The two other S. typhosa Hfr hybrids, on the basis of transmission frequency gradients, appeared to contain a continuous E. coli diploid segment complete from proA through the fuc locus. Other classes of S. typhosa Hfr hybrids, derived from mating with E. coli Hfr WR2010 (O...tna...xyl), were also observed to transfer E. coli genes at high frequency.     

Inefficiency of genetic recombination in hybrids between Escherichia coli and Salmonella typhosa

An Escherichia coli Hfr strain in which three negative chromosomal alleles (leu(-), arg(-), and mtl(-)) were closely linked to three positive alleles (ara(+), rha(+), and xyl(+), respectively) was employed in matings with a Salmonella typhosa recipient. The detected expression of the negative E. coli alleles in S. typhosa hybrids selected for receipt of an associated positive E. coli marker was used to determine the occurrence of haploid S. typhosa recombinants, as distinguished from stable partial diploid hybrids. At the same time, the inheritance patterns and segregation behavior of the positive alleles provided indicators of the occurrence of partial diploid hybrids. Examination of both positive and negative markers inherited by ara(+), rha(+), and xyl(-) selected S. typhosa hybrid classes indicated that relatively short E. coli chromosomal segments (generally about 4 min or less in length) were involved in recombination (haploidy), whereas rather extensive E. coli genetic segments were conserved in the diploid state. S. typhosa hybrids selected for receipt of the ara(+) marker showed a 52% incidence of leu(-) haploidy, which is probably close to being an accurate measure of recombination at the site of the ara(+) allele. S. typhosa hybrids selected for receipt of the rha(+) or xyl(+) markers showed only a 20% incidence of arg(-) or mtl(-) haploidy, respectively, but both of these hybrid classes exhibited a higher incidence of conservation of extensive E. coli diploid segments than did the ara(+) selected class. Remating of haploid S. typhosa hybrids with recombinant xyl(+)mtl(-) or rha(+)arg(-) regions resulted in higher frequencies of hybrid recovery than were observed in the initial matings. However, there was a higher incidence of partial diploidy and a lower incidence of haploidy among the hybrids obtained from these rematings.     

Construction of intergeneric hybrids using bacteriophage P1CM: transfer of the Klebsiella aerogenes ribitol dehydrogenase gene to Escherichia coli.

Study of many of the interesting properties of Klebsiella aerogenes is limited by the lack of a well-characterized genetic system for this organism. Our investigations of the evolution of the enzyme ribitol dehydrogenase (EC 1.1.1.56) in K. aerogenes would be greatly facilitated by the availability of such a system, and we here report two approaches to developing one. We have isolated mutants sensitive to the coliphage P1, which will efficiently tranduce genetic markers between such sensitive strains and which will thus make detailed mapping studies possible. Derivatives of K. aerogenes lysogenic for P1 can be readily isolated by using the specialized transducing particle P1CMclr100. Bacteria lysogenic for this phage are chloramphenicol resistant and temperature sensitive. Phage particles produced by temperature induction of such lysogens can be used to transfer K. aerogenes genes to the natural host of P1 phage. Escherichia coli. We have used this method to prepare derivatives of E. coli K-12 carrying the K. aerogenes genes conferring the ability to metabolize the pentitols ribitol and D-arabitol. We have shown that these E. coli-K. aerogenes hybrids synthesize a ribitol dehydrogenase with the properties of the K. aerogenes enzyme and have mapped the position of the transferred gene on the E. coli chromosome. The ramifications of this methodology are discussed.     

Intergeneric conjugational hybridization of Escherichia coli and Salmonella typhimurium. 1. Obtaining a salmonella hybrid possessing greater recipient activity in crosses with Escherichia coli]

Intergeneric hybrids were selected from mating HfrH Escherichia coli with F- Salmonella typhimurium. The hybrid obtained from E. coli leu+ and pro+ genes possessed the increased recipient ability in the mating with E. coli HfrR1 (O--ilv--metE--ara). This hybrid lacked the ability to restrict the phage P1 DNA propagated on E. coli K-12. The replacement of mutated uvrA gene of Salmonella for uvrA+ gene of E. coli restore uvr+ phenotype of Salmonella mutant.     

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.