New derivatives of transposon tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in Gram-negative bacteria

Simon, Reinhard; Quandt, Jürgen; Klipp, Werner

doi:10.1016/0378-1119(89)90262-x

Cited by 331 publications

(181 citation statements)

References 42 publications

Supporting

Mentioning

173

Contrasting

Unclassified

Order By: Relevance

“…Strain 3841 was mutagenized with Tn5 by conjugation with E. coli S17-1 Phagemid, pUC19 derivative, f l origin of replication, ColEl pACY C derivative, P15A origin of replication, GmR IncP transcriptional fusion vector, TcR P-group chaser plasmid, GmR pLAFRl cosmid containing mdh-sucCDAB from strain 3841 pLAFRl cosmid containing aapJQMP from strain 3841 pRU3024 aapJ : : Tn5-facZ pRU3004 sucC : : Tn5-facZ pRU3004 sucA : : Tn5-lac2 pRU3004 sucA : : Tn5-lac2 pRU3004 sucA : : Tn5-lac2 pRU3004 sucB : : Tn5-facZ pRU3004 rndh : : Tn5-lacZ pRU3004 sucA : : Tn5-facZ pRU3004 mdh : : Tn5-lac2 Promoterless cloning vector, AmpR mob KmR replicon, AmpR Buchanan-Mollaston (1979) containing pSUP202-1: : Tn5 essentially as described by Simon et al (1983). Cosmid pRU3024 was mutagenized with Tn5-lacZ by using 1 containing the transposon derivative B20 essentially as described by Simon et al (1989). To create chromosomal mutations, mutated cosmids were conjugated into R. legurninosarum strain 3841.…”

Section: Methodsmentioning

confidence: 99%

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

et al. 1997

View full text Add to dashboard Cite

Reading RG6 6AJ, UKMutants of Rhizobium leguminosarum were selected that were altered in the uptake activity of the general amino acid permease (Aap). The main class of mutant maps to s u d and suct), which are part of a gene cluster mdh-sucCDAB, which codes for malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD) and components of the 2-oxoglutarate dehydrogenase complex ( s u d B ) . Mutation of either SUCC or SUCD prevents expression of 2-oxoglutarate dehydrogenase (sucAB). Conversely, mutation of sucA or sucB results in much higher levels of succinyl-CoA synthetase and malate dehydrogenase activity. These results suggest that the genes mdh-sucCDAB may constitute an operon. suc mutants, unlike the wild-type, excrete large quantities of glutamate and 2-oxoglutarate. Concomitant with mutation of s u d or suct), the intracellular concentration of glutamate but not 2-oxoglutarate was highly elevated, suggesting that 2-oxoglutarate normally feeds into the glutamate pool. Elevation of the intracellular glutamate pool appeared to be coupled to glutamate excretion as part of an overflow pathway for regulation of the TCA cycle. Amino acid uptake via the Aap of R. leguminosamm was strongly inhibited in the suc mutants, even though the transcription level of the aap operon was the same as the wild-type. This is consistent with previous observations that the Aap, which influences glutamate excretion in R. leguminosarum, has uptake inhibited when excretion occurs. Another class of mutant impaired in uptake by the Aap is mutated in polyhydroxybutyrate synthase (phaC). Mutants of succinyl-CoA synthetase (SUCD) or 2-oxoglutarate dehydrogenase (sucA) form ineffective nodules. However, mutants of aap, which are unable to grow on glutamate as a carbon source in laboratory culture, show wild-type levels of nitrogen fixation. This indicates that glutamate is not an important carbon and energy source in the bacteroid. Instead glutamate synthesis, like polyhydroxybutyrate synthesis, appears to be a sink for carbon and reductant, formed when the 2-oxoglutarate dehydrogenase complex is blocked. This is in accord with previous observations that bacteroids synthesize high concentrations of glutamate. Overall the data show that the TCA cycle in R. leguminosarum is regulated by amino acid excretion and polyhydroxybutyrate biosynthesis which act as overflow pathways for excess carbon and reductant.

show abstract

Section: Methodsmentioning

confidence: 99%

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

et al. 1997

View full text Add to dashboard Cite

show abstract

“…To isolate strains defective in biofilm formation on an abiotic surface, Tn5-based transposons that confer Tc r or Gm r (Simon et al, 1989) were used to mutagenize P. fluorescens. Of the approximately 14 000 transposon mutants screened, 37 mutants (0.3%) were unable to form a biofilm ( Fig.…”

Section: Isolation Of Mutants Defective In Biofilm Formationmentioning

confidence: 99%

Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis

O’Toole

Kolter

1998

Molecular Microbiology

2,258

1,795

View full text Add to dashboard Cite

SummaryPopulations of surface-attached microorganisms comprising either single or multiple species are commonly referred to as biofilms. Using a simple assay for the initiation of biofilm formation (e.g. attachment to an abiotic surface) by Pseudomonas fluorescens strain WCS365, we have shown that: (i) P. fluorescens can form biofilms on an abiotic surface when grown on a range of nutrients; (ii) protein synthesis is required for the early events of biofilm formation; (iii) one (or more) extracytoplasmic protein plays a role in interactions with an abiotic surface; (iv) the osmolarity of the medium affects the ability of the cell to form biofilms. We have isolated transposon mutants defective for the initiation of biofilm formation, which we term surface attachment defective (sad ). Molecular analysis of the sad mutants revealed that the ClpP protein (a component of the cytoplasmic Clp protease) participates in biofilm formation in this organism. Our genetic analyses suggest that biofilm formation can proceed via multiple, convergent signalling pathways, which are regulated by various environmental signals. Finally, of the 24 sad mutants analysed in this study, only three had defects in genes of known function. This result suggests that our screen is uncovering novel aspects of bacterial physiology.

show abstract

“…As part of an ongoing interest in S. meliloti functional genomics and carbon catabolism pathways that may play a role in rhizosphere survival, strain RmG212, a Lac 2 derivative of Rm1021 (Glazebrook & Walker, 1991), was subjected to random mutagenesis with the transposon Tn5-B20 that is capable of generating transcriptional lacZ fusions (Simon et al, 1989). Mutants containing transposon insertions were screened for a lack of ability to grow on a number of sugars as a sole carbon source, including arabinose.…”

Section: Isolation Of Arabinose Catabolism Mutantsmentioning

confidence: 99%

Sinorhizobium meliloti pSymB carries genes necessary for arabinose transport and catabolism

et al. 2007

View full text Add to dashboard Cite

Arabinose is a known component of plant cell walls and is found in the rhizosphere. In this work, a previously undeleted region of the megaplasmid pSymB was identified as encoding genes necessary for arabinose catabolism, by Tn5-B20 random mutagenesis and subsequent complementation. Transcription of this region was measured by b-galactosidase assays of Tn5-B20 fusions, and shown to be strongly inducible by arabinose, and moderately so by galactose and seed exudate. Accumulation of [ 3 H]arabinose in mutants and wild-type was measured, and the results suggested that this operon is necessary for arabinose transport. Although catabolite repression of the arabinose genes by succinate or glucose was not detected at the level of transcription, both glucose and galactose were found to inhibit accumulation of arabinose when present in excess. To determine if glucose was also taken up by the arabinose transport proteins, [ 14 C]glucose uptake rates were measured in wild-type and arabinose mutant strains. No differences in glucose uptake rates were detected between wild-type and arabinose catabolism mutant strains, indicating that excess glucose did not compete with arabinose for transport by the same system. Arabinose mutants were tested for the ability to form nitrogen-fixing nodules on alfalfa, and to compete with the wild-type for nodule occupancy. Strains unable to utilize arabinose did not display any symbiotic defects, and were not found to be less competitive than wild-type for nodule occupancy in co-inoculation experiments. Moreover, the results suggest that other loci are required for arabinose catabolism, including a gene encoding arabinose dehydrogenase.

show abstract

New derivatives of transposon tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in Gram-negative bacteria

Cited by 331 publications

References 42 publications

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum

Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis

Sinorhizobium meliloti pSymB carries genes necessary for arabinose transport and catabolism

Contact Info

Product

Resources

About