Specialized Transducing Phages Derived from Salmonella Phage P22

Salmonella phage P22 has been used in the construction of three sorts of specialized transducing phage: P22 proAB, P22 proABlac and P22 argF. The bacterial genes carried are derived from E. coli K12. Since E. coli and Salmonella chromosomes recombine very poorly, E. coli genes cannot be transduced into Salmonella recipients by P22's generalized transduction mechanism. Therefore, stable inheritance of E. coli material provides a means of detecting specialized transduction. Formation of these phages was possible because the P22 prophage recognizes an attachment site in the E. coli F' prolac episome. Salmonella strains carrying the F' prolac episome can be lysogenized by P22 so as to leave the prophage inserted into the E. coli material of the F' factor. Improper prophage excision can then lead to formation of P22 specialized phages carrying E. coli genetic material.     

Sub-cloning of the wild-type proAB region of the Escherichia coli genome

The genes proA and proB encoding the first two enzymes of the proline biosynthetic sequence in Escherichia coli were subcloned from a ColE1 hybrid plasmid containing 23.3 kilobases of genomic DNA. proA and proB are contiguous and constitute a single operon transcribed in the direction proB-proA. The pro operon is contiguous with the gene phoE. Hybridization experiments showed no homology between proAB of E. coli and the other regions of the E. coli genome or with the DNA of several other bacterial species.     

A specialised transducing phage of P22 for which the ability to form plagues is associated with transduction of the proAB region

Summary The multisite mutantproAB47 has been used to isolate specialised transducing phages of P22 for theproA andproB genes. Several of the preparations contain phages that form small plaques, and many of the properties of these suggest that the small plaques are formed by the transducing phages. Preliminary studies on the transduction of theproAB region by one such phage suggest that it forms fairly stable heterogenotes, both from apro point mutant and fromproAB47. The fact that the latter mutant is missing the phage attachment site results in a delay in integration of the specialised transducing phage.     

Cloning of genes for proline biosynthesis in Escherichia coli

Restriction map of Escherichia coli chromosome fragment (7.4 MD) carrying proAB genes was constructed. Localization of proA and proB genes on the cloned chromosome fragment was determined by complementation test and the measuring of glutamylkinase activity (proB gene product). ProA and proB genes were cloned separately on multicopy plasmids of alternative orientation and their expression being, probably, under the control of their own regulatory regions, studied.     

Genetic Separation of Hypoxanthine and Guanine-Xanthine Phosphoribosyltransferase Activities by Deletion Mutations in Salmonella typhimurium

Certain proAB deletion mutants of Salmonella typhimurium were found to be simultaneously deleted in a gene required for the utilization of guanine and xanthine (designated gxu). These mutants were resistant to 8-azaguanine and when carrying an additional pur mutation were unable to use guanine or xanthine as a purine source. The defect was correlated with deficiencies in the uptake and phosphoribosyltransferase activities for guanine and xanthine. Hypoxanthine and adenine activities were unaltered. The deficiency was restored to normal by transduction to pro(+) and in F' merodiploids.     

Regulation of purine utilization in bacteria. VI. Characterization of hypoxanthine and guanine uptake into isolated membrane vesicles from Salmonella typhimurium.

Uptake of hypoxanthine and guanine into isolated membrane vesicles of Salmonella typhimurium TR119 was stimulated by 5'-phosphoribosyl-1'-pyrophosphate (PRPP). For strain proAB47, a mutant that lacks guanine phosphoribosyltransferase, PRPP stimulated uptake of hypoxanthine into membrane vesicles. No PRPP-stimulated uptake of guanine was observed. For strain TR119, guanosine 5'-monophosphate and inosine 5'-monophosphate accumulated intravesicularly when guanine and hypoxanthine, respectively, were used with PRPP as transport substrates. For strain proAB47, IMP accumulated intravesicularly with hypoxanthine and PRPP as transport substrates. For strain TR119, hypoxanthine also accumulated when PRPP was absent. This free hypoxanthine uptake was completely inhibited by N-ethylmaleimide, but the PRPP-stimulated uptake of hypoxanthine was inhibited only 20% by N-ethylmaleimide. Hypoxanthine and guanine phosphoribosyltransferase activity paralleled uptake activity in both strains. But, when proAB47 vesicles were sonically treated to release the enzymes, a three- to sixfold activation of phosphoribosyltransferase molecules occurred. Since proAB47 vessicles lack the guanine phsophoribosyltransferase gene product and since hypoxanthine effectively competes out the phosphoribosylation of guanine by proAB47 vesicles, it was postulated that the hypoxanthine phosphoribosyltransferase gains specificity for both guanine and hypoxanthine when released from the membrane. A group translocation as the major mechanism for the uptake of guanine and hypoxanthine was proposed.     

Recombination in Escherichia coli cells and multiform cytotoxic resistance

Genetic crosses between E. coli Hfr C--donor and E. coli AB 1157--recipient cells sensitive to single target inhibitor glyphosate gave rise to offspring which could tolerate not only minimal inhibitory concentration of this inhibitor but also six-fold increased concentrations. No change in glyphosate target enzyme activity in transconjugants (resulted from recombination occurred at arg G and proAB genome regions, respectively) compared to that of parent strains was observed. If new combinations due to recombination of some genetic factors is responsible for emergence of this resistance the question is raised on the nature of these factors and their role in cell biology.     

Stable shuttle vectors for Neisseria gonorrhoeae, Haemophilus spp. and other bacteria based on a single origin of replication

An origin of replication (ori) was obtained from a naturally occurring beta-lactamase-producing plasmid isolated from Neisseria gonorrhoeae and used to construct shuttle vectors capable of replicating in N. gonorrhoeae, Haemophilus ducreyi, Haemophilus influenzae and Escherichia coli. Using the gonococcal proAB genes, we complemented proline-requiring N. gonorrhoeae F62 and E. coli HB101 in trans. The first demonstration of the expression of the green fluorescent protein (GFP) in either N. gonorrhoeae or H. ducreyi was shown using this vector, indicating that GFP may be a useful tool in the analysis of these organisms. This is the first report of a gonococcal vector based on a broad host range, genetically defined ori, and should facilitate the molecular analysis of gonococcal and Haemophilus genes.     

Inversion in the lactose region of Escherichia coli K-12.

A spontaneous mutant of Escherichia coli K-12, strain SY99, with an inversion in the lactose region was isolated and partially characterized. The inversion was detected due to inverse chromosomal conjugational transfer after introduction of an F42 (F'lac) episome. The termini of the inversion are between proAB and lac on one side and lac and proC on the other. The inverse conjugational transfer in SY99 did not appear to be absolute but was always accompanied by a residual "normal" counterclockwise mobilization. This residual transfer was further shown to be caused by the intrinsic instability of this region (at least in the line W3110). The possible involvement of IS3 elements flanking the lactose operon is discussed.     

Construction of an L-arginine-producing mutant in Serratia marcescens. Use of the wide substrate specificity of acetylornithinase.

L-Arginine biosynthesis in Serratia marcescens Sr41 was found to be controlled by (a) feedback inhibition of N-acetylglutamate synthetase and (b) repression of some L-arginine biosynthetic enzymes, and an L-arginine-degrading system was found to exist. Accordingly, an L-arginine-producing mutant (aru argR argA) of S. marcescens Sr41 was constructed as follows. A mutant incapable of L-arginine utilization (aru) was obtained from the wild strain. Subsequently, from the lysine auxotroph (lysA) of aru mutant, a mutant having derepressed L-arginine biosynthetic enzymes (argR) was isolated by screening for colonies that could utilize Nalpha-acetyl-L-lysine in the presence of L-arginine. This selection was based on the finding that acetylornithinase of S. marcescens hydrolyzed Nalpha-acetyl-L-lysine. On the other hand, to obtain a mutant with feedback-resistant N-acetylglutamate synthetase (argA), the proAB argD argR triple mutant was isolated from the indirectly suppressed revertant (proAB argD) of the proline auxotroph (proAB). Next, the argA mutant was isolated from the triple mutant by selection for resistance to 3,4-dehydro-DL-proline in the presence of L-arginine. The argA mutation was introduced into the aru lysA argR strain by PS20-mediated cotransduction with lysA+. The aru argR argA lysA+ transductant produced 25 mg/ml of L-arginine in the medium.     

Cloning genes for proline biosynthesis from Neisseria gonorrhoeae: identification by interspecific complementation of Escherichia coli mutants

DNA from Neisseria gonorrhoeae KH45 was partially digested with Sau3A and inserted into the BamHI site of the cloning vector pLES2 . After introduction into Escherichia coli JM83 by transformation, two different size classes of plasmids were isolated that could complement the proAB deletion of JM83 . These plasmids ( pLES4 and pLES7 ) were characterized by restriction endonuclease digestion. Southern hybridization demonstrated that the inserts had sequence homology. Various deletions of these plasmids were constructed that had lost the ability to complement the proA lesion of chi 463, the proB lesion of chi 340, or both (plasmids pLES9 , pLES8 , and pLES10 , respectively). These deleted plasmids were introduced into a proline-requiring strain of N. gonorrhoeae, F62, with plasmids pLES4 , pLES7 , and pLES8 possessing the ability to correct the proline requirement of F62. Further analysis indicated that the hybrid plasmids were stably maintained as plasmids in N. gonorrhoeae.     

Endonuclease A degrades chromosomal and plasmid DNA of Escherichia coli present in most preparations of single stranded DNA from phagemids.

With E. coli, large and variable amounts of chromosomal and plasmid DNAs are observed in the supernatants of overnight cultures when the cells carry an endA mutation, but are not detected by gel electrophoresis when the cells carry the wild type allele of endA. Significant amounts of nuclease activity in DH11S endA+ supernatants were detected by two simple assays; the rapid degradation of added pBR322 plasmid DNA, as judged by agarose gel electrophoresis, and a decrease of more than 100000 fold in transformation efficiency of the added pBR322 plasmid DNA. By employing isogenic endA mutant and wild type strains of DH11S and DH10B/F' proAB+ laclq Z delta M15, it was shown that detectable levels of chromosomal and plasmid DNAs are observed only in the endA mutant strains. These results indicate that Endonuclease I activity is responsible for degradation of chromosomal and plasmid DNA usually present in preparations of ssDNA. Therefore, a wild type endA gene is useful for the rapid and simple production of highly purified ssDNA from cells containing phagemid vectors.     

Suppression of Proline Requirement of proA and proAB Deletion Mutants in Salmonella typhimurium by Mutation to Arginine Requirement

Eleven variants able to grow without proline (provided arginine was absent) were obtained by spontaneous mutation from Salmonella typhimurium LT7 proA and proAB deletion mutants. Suppression resulted from mutation at argG, which specifies N(alpha)-acetylornithine delta-transaminase. In the absence of exogenous arginine, deficiency of this enzyme would cause derepression of the arginine pathway and accumulation of N-acetylglutamic gamma-semialdehyde. N-acetylglutamic gamma-semialdehyde, if deacetylated, would produce glutamic gamma-semialdehyde, the proline precursor whose synthesis from glutamate is blocked in proA and proAB mutants. All of the mutants grew only slowly (some very slowly) if not supplied with arginine. Sonic-treated preparations of eight mutants had no measurable acetylornithine delta-transaminase activity, but those of the three mutants least dependent on arginine had 0.11, 0.28, and 1.48 of wild-type activity; presumably, their enzymes have low specific activity, at least in vivo. Phage P22 cotransduced argG and strA. Genetic analysis showed that the minor degree of arginine dependence of the mutant with greater than wild-type in vitro enzyme activity was a characteristic of its argG allele, not the result of modification of the argG phenotype by mutation elsewhere.     

Proline excretion and indirect suppression in Escherichia coli and Salmonella typhimurium

The last step in proline biosynthesis in Escherichia coli K-12, Salmonella typhimurium LT7, and a number of other enterobacterial isolates is regulated so that no proline is excreted, even if excess Delta(1)-pyrroline-5-carboxylate, the immediate precursor of proline, is added to a culture. In proline auxotrophs blocked at an early step in proline biosynthesis (proA or proB), reversion to prototrophy is often due to a mutation in the arginine pathway which diverts N-acetyl glutamate gamma-semialdehyde to proline synthesis, thus bypassing the proA or proB block. In such double mutants (proAB, argD), the last step in proline synthesis appears to be unregulated, since proline is excreted. Feedback inhibition and repression of the arginine pathway overcomes indirect suppression (restoring the Pro(-) phenotype), but proline regulation is not restored; double mutants still excrete proline when fed Delta(1)-pyrroline-5-carboxylate exogeneously. A new class of proline analogue-resistant mutant, due to mutation at argD, is also described.     

Transduction of chromosomal genes between enteric bacteria by bacteriophage P1.

We have used P1 transduction to create intergeneric hybrid strains of enteric bacteria by moving the genA and hut genes between Klebsiella aerogenes, Escherichia coli and Salmonella typhimurium. The use of E. coli as the recipient in such transductions permits the construction of episomes and specialized transducing phage containing non-E. coli material. The effect of host restriction modification and deoxyribonucleic acid homology on the frequency of intergeneric transduction of these loci has been examined.     

Escherichia coli strain with a deletion of the chromosomalampC gene marked with TcR, suitable for production of penicillin G acylase

Escherichia coli strain which contains a marker of tetracycline resistance gene (TcR) placed by P1 transduction beside the chromosomal deletion of ampC gene (delta ampC) coding for beta-lactamase was constructed. Such introduction of TcR marker permits a fast and simple selection for the transfer of delta ampC by P1 transduction into industrial E. coli strains. This approach was used for constructing an E. coli strain suitable for penicillin acylase production.     

Biochemical characterization of a mutant of the yeast Pichia anomala derepressed for malic acid utilization in the presence of glucose

Abstract Myxococcus xanthus cells move over surfaces by gliding motility. The frz signal transduction system is used to control the reversal frequency, and thus the overall direction of movement of M. xanthus cells. We analyzed the behavior of wild-type and frz mutant cells in response to prey bacteria ( Escherichia coli ). Wild-type cells of M. xanthus did not respond to microcolonies of E. coli until they made physical contact. Cells which penetrated a colony remained in the colony until all of the prey cells were digested. Cells of frz mutants also penetrated E. coli microcolonies and digested some of the E. coli cells, but they invariably abandoned the microcolony leaving their food source behind. These observations illustrate the importance of the frz system of signal transduction for the feeding behavior of M. xanthus cells.     

Involvement of the AtoS-AtoC signal transduction system in poly-(R)-3-hydroxybutyrate biosynthesis in Escherichia coli

The AtoS-AtoC signal transduction system in E. coli, which induces the atoDAEB operon for the growth of E. coli in short-chain fatty acids, can positively modulate the levels of poly-(R)-3-hydroxybutyrate (cPHB) biosynthesis, a biopolymer with many physiological roles in E. coli. Increased amounts of cPHB were synthesized in E. coli upon exposure of the cells to acetoacetate, the inducer of the AtoS-AtoC two-component system. While E. coli that overproduce both components of the signal transduction system synthesize higher quantities of cPHB (1.5-4.5 fold), those that overproduce either AtoS or AtoC alone do not display such a phenotype. Lack of enhanced cPHB production was also observed in cells overexpressing AtoS and phosphorylation-impaired AtoC mutants. The results were not affected by the nature of the carbon source used, i.e., glucose, acetate or acetoacetate. An E. coli strain with a deletion in the atoS-atoC locus (delta atoSC) synthesized lower amounts of cPHB compared to wild-type cells. When the delta atoSC strain was transformed with a plasmid carrying a 6.4-kb fragment encoding the AtoS-AtoC system, cPHB biosynthesis was restored to the level of the atoSC+ cells. Introduction of a multicopy plasmid carrying a functional atoDAEB operon, but not one with a promoterless operon, resulted in increased cPHB synthesis only in atoSC+ cells in the presence of acetoacetate. These results indicate that the presence of both a functional AtoS-AtoC two-component signal transduction system and a functional atoDAEB operon is critical for the enhanced cPHB biosynthesis in E. coli.     

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.     

Physical and genetical analysis of bacteriophage T4 generalized transduction

This report describes a comparison of the efficiency of transduction of genes in E. coli by the generalized transducing bacteriophages T4GT7 and P1CM. Both phages are capable of transducing many genetic markers in E. coli although the frequency of transduction for particular genes varies over a wide range. The frequency of transduction for most genes depends on which transducing phage is used as well as on the donor and recipient bacterial strains. Analysis of T4GT7 phage lysates by cesium chloride density gradient centrifugation shows that transducing phage particles contain primarily bacterial DNA and carry little, if any, phage DNA. In this regard transducing phages P1CM and T4GT7 are similar; both phages package either bacterial or phage DNA but not both DNAs into the same particle.