Isolation of ara-lac gene fusions in Salmonella typhimurium LT2 by using transducing bacteriophage Mu d (Apr lac)

Lee, J H; Heffernan, L; Wilcox, Gary

doi:10.1128/jb.143.3.1325-1331.1980

Cited by 31 publications

(19 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A method is described to convert existing Mu dl insertions to Mu dX. The Mu dl (lac Apr) phage (5) is a powerful tool for the genetic dissection of complex regulatory pathways in bacteria (12)(13)(14)18). Mu dl insertions are essentially random and are marked by an Apr (ampicillin resistance) gene which facilitates both their selection and mapping.…”

mentioning

confidence: 99%

Mu dX, a derivative of Mu d1 (lac Apr) which makes stable lacZ fusions at high temperature

Baker¹,

Howe²,

Gross³

1983

J Bacteriol

View full text Add to dashboard Cite

We describe defective Mu phage Mu dX (Mu dl BX::Tn9 [lac Apr Cmr]) which is useful for insertion mutagenesis and for construction of lac operon fusions in vivo. Mu dX retains the insertion properties of Mu dl but produces temperatureresistant lysogens and transposes at a reduced frequency. A method is described to convert existing Mu dl insertions to Mu dX. The Mu dl (lac Apr) phage (5) is a powerful tool for the genetic dissection of complex regulatory pathways in bacteria (12)(13)(14)18). Mu dl insertions are essentially random and are marked by an Apr (ampicillin resistance) gene which facilitates both their selection and mapping. In addition, insertions in one orientation place the lacZ and lacY genes under the control of the promoter of the gene carrying the insertion. Thus, the regulatory properties of an operon can be investigated by measuring ,-galactosidase expression from the operon fusion. There are, however, two features of the Mu dl phage which limit its utility. First, Mu dl lysogens are temperature sensitive for growth due to inactivation of the cts repressor and consequent induction of Mu replication and kil function at high temperature. Second, Mu dl insertions are genetically unstable since secondary Mu-specific replicative transpositions occur at a relatively high frequency. This latter feature of Mu dl makes it impractical to use genetic selections based on the Mu dl operon fusion phenotype (8) and has necessitated the development of methods for stabilizing Mu dl fusions after their isolation (13).Previous studies have established that polar insertions in the Mu B gene (X mutations) prevent the expression of both B and kil and thus eliminate the temperature-sensitive growth phenotype of a Mu cts lysogen (2). Thus, we expected that an X mutation should eliminate the temperature sensitivity of a Mu dl lysogen. The X mutation should also reduce the frequency of secondary transposition because the B gene product is required for Mu-specific DNA replication (7). We constructed a Mu dl phage carrying a Bx mutation caused by insertion of Tn9 (chloramphenicol-resistant, Cm9 into the Mu B t Present address:

show abstract

mentioning

confidence: 99%

Mu dX, a derivative of Mu d1 (lac Apr) which makes stable lacZ fusions at high temperature

Baker¹,

Howe²,

Gross³

1983

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…The regulation of araC is different from that of araBAD (7), suggesting araC is not part of the araBAD transcription unit. To determine the transcription direction of araC, an araC-lac fusion strain, L1236, was used.…”

mentioning

confidence: 87%

“…If araC in S. typhimurium is also transcribed clockwise, it should be possible to isolate deletions that extend into leu from an araC-lac fusion strain. Strain L1236, which contains a temperature-sensitive Mu d(Apr lac) phage in the araC gene (7), was heat induced as described (6), and the ampicillin-resistant survivors were screened for the Leu phenotype. Two Lac' Leu-Apr colonies were obtained from approximately 2,000 colonies, suggesting that the transcription direction of araC in S. typhimurium is the same as in E. coli.…”

mentioning

confidence: 99%

Genetic characterization of Salmonella typhimurium LT2 ara mutations

Lee¹,

Nishitani²,

Wilcox³

1984

J Bacteriol

Self Cite

View full text Add to dashboard Cite

Seventeen independently isolated L-arabinose utilization-deficient mutants of Salmonella typhimurium LT2 were characterized. Four complementation groups (araA, araB, araC, and araD) were identified and were equivalent to the same genes in the ara system in Escherichia coli. The order of the four genes was determined to be araD-araA-araB-araC-leu. Two transcription units were found: the araBAD operon was transcribed counterclockwise, and the araC gene was transcribed clockwise.Although the genetic organization of the ara system in Escherichia coli has been extensively studied (for reviews, see references 2 and 9), less information concerning ara gene organization in Salmonella typhimurium is available. Mutations for defective L-arabinose utilization have been located at three units, between fol and leu, on the S. typhimurium genetic map (5). The transport genes have been studied in both E. coli and S. typhimurium with the surprising observation that there is no functional araF in S. typhimurium (8). In this work, we further investigated the L-arabinose gene complex in S. typhimurium.Characterization of ara mutants. The bacterial strains used and their origins are listed in Table 1. Twenty-three ara mutants isolated from N-methyl-N'-nitro-N-nitrosoguanidine-mutagenized LOO1 cells were mapped with the generalized transducing phage P22. All were cotransducible with leu. The cotransduction frequencies were between 25 and 40% when TT206, a leu: :TnJO strain, was used as the recipient (data not shown). Among the 23 mutants, 6 were leaky and were not studied further. The other 17 mutants were characterized in more detail.Since S. typhimurium and E. coli are closely related, the ara genes may be able to complement each other. Strains of E. coli carrying ara mutations on F' factors were available. The complementation tests were performed with E. coli B/r F' strains used as donors. The experiments were not successful, probably because of the restriction barrier between E. coli and S. typhimurium. To overcome this problem, four E. coli F' factors, each carrying a mutation in araA, araB, araC, or araD, were transferred into a restriction-deficient, modification-proficient S. typhimurium strain, L0603. The constructed F' strains (L0606 through L0609) were then used as donors in the complementation test. Two ara mutants which were L-arabinose sensitive, L727 and L6112, were also analyzed by complementation. The mutants fell into four complementation groups: two in araC, nine in araB (of which four were polar on araA and araD), four in araA (of which two were polar on araD), and three in araD. The reversion frequency of the noncomplemented strain, L6014, * Corresponding author. t Present address: INGENE, 1701 Colorado Ave., Santa Monica, CA 90404.

show abstract

“…Csonka et al (15) developed a procedure for introducing the Mu dl phage into Salmonella typhimurium, thereby increasing the host range of this phage. The Mu phage can also be introduced into other gram-negative bacteria that can receive plasmids containing an insertion of a Mu prophage (16,25,28). By using Mu dl and Mu d2 prophages inserted into the broad-host-range R-factor R751 and crossing it from E. coli to other gram-negative species, we thought to further enlarge the host range of these genetic tools.…”

mentioning

confidence: 99%

In vivo formation of gene fusions in Pseudomonas putida and construction of versatile broad-host-range vectors for direct subcloning of Mu d1 and Mu d2 fusions

Simon

Schumann

1987

Appl Environ Microbiol

View full text Add to dashboard Cite

The Mu dl and Mu d2 prophages were integrated into the conjugative broad-host-range plasmid R751. The two plasmids were then transferred into Pseudomonas putida, and derivatives carrying intact Mu prophages were recovered. After induction of Mu at 42°C, both operon and gene fusions were observed on 5-bromo-4chloro-3-indolyl-i-D-galactopyranoside (X-Gal) plates. Broad-host-range vectors were constructed which allow direct cloning of both operon or gene fusions and their analysis in Escherichia coli and P. putida. By using one of these vectors, two operon fusions were isolated from the P. putida chromosome and comparatively analyzed in E. coli and P. putida.

show abstract

Isolation of ara-lac gene fusions in Salmonella typhimurium LT2 by using transducing bacteriophage Mu d (Apr lac)

Cited by 31 publications

References 21 publications

Mu dX, a derivative of Mu d1 (lac Apr) which makes stable lacZ fusions at high temperature

Mu dX, a derivative of Mu d1 (lac Apr) which makes stable lacZ fusions at high temperature

Genetic characterization of Salmonella typhimurium LT2 ara mutations

In vivo formation of gene fusions in Pseudomonas putida and construction of versatile broad-host-range vectors for direct subcloning of Mu d1 and Mu d2 fusions

Contact Info

Product

Resources

About