Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria The GenBank accession numbers for the sequences reported in this paper are AF327711, AF327712, AF327713, AF327714, AF327715, AF327716, AF327717, AF327718, AF327719 and AF327720.

Abstract

Full exploitation of the information available in bacterial genome sequences requires the availability of facile tools for rapid genetic manipulation. One bacterium for which new genetic tools are needed is the methylotroph Methylobacterium extorquens AM1. IncQ and small IncP vectors were shown to be unsuitable for use in this bacterium, but a spontaneous mutant of a small IncP plasmid was isolated that functioned efficiently in M. extorquens AM1. This plasmid was sequenced and used as a base for developing improved broad-host-range cloning vectors. These vectors were found to replicate in a wide variety of bacterial species and have the following advantages : (1) high copy number in Escherichia coli ; (2) small size (72 and 80 kb) ; (3) complete sequences ; (4) variety of unique restriction sites ; (5) blue-white screening via lacZα ; (6) conjugative mobilization between bacterial species ; and (7) readily adaptable into species-specific promoter-probe and expression vectors. Two low-background promoter-probe vectors were constructed based on these cloning vectors with either lacZ or xylE as reporter genes ; these were shown to report gene expression effectively in M. extorquens AM1. Specific expression vectors were developed for use in M. extorquens AM1, which were shown to express foreign genes at significant levels, and a simple strategy is outlined to develop specific expression vectors for other bacteria. The strong mxaF promoter was used for expression, since E. coli lac-derived promoters were expressed at very low levels. This suite of genetic tools will enable a more sophisticated analysis of the physiology of M. extorquens AM1, and these vectors should also be valuable tools in the study of a variety of bacterial species.

Reconstruction of C3 and C4 metabolism in Methylobacterium extorquens AM1 using transposon mutagenesis

“…Genome sequence data obtained as described above were used to design PCR primers for the amplification of the wild-type gene. Products of the expected sizes were obtained and isolated, cloned directly into pCR2.1 and subcloned downstream of the P mxaF promoter in the M. extorquens AM1 expression vector pCM80 (Marx & Lidstrom, 2001) as either EcoRI-EcoRI or XbaI-KpnI fragments.…”

“…The recent availability of the M. extorquens AM1 genome sequence along with the advent of genetic tools for the construction of insertion mutants (Chistoserdov et al, 1994) and the overexpression of genes (Marx & Lidstrom, 2001) now enable the application of genomic approaches to the understanding of metabolic pathways in this organism. In a previous study, five genes predicted to be involved in growth on succinate or pyruvate encoding citrate synthase, succinate dehydrogenase, malic enzyme, phosphoenolpyruvate synthase and phosphoenolpyruvate carboxykinase were identified in the genome sequence.…”

Heat-shock sigma factor RpoH from Geobacter sulfurreducens

“…The spectinomycin-resistance gene was amplified with pSJS985Q (Sandler & Clark, 1994) as a template and primers 59-TCGAATTCACAGGATGACGCCTAAC-39 and 59-TC-CTCGAGTCTAACGCTTGAGTTAA-39 (EcoRI and XhoI sites are underlined). The HindIII-EcoRI fragment of the rpoH gene and the EcoRI-XhoI fragment of the spectinomycin-resistance gene were cloned into the XhoI-HindIII fragment from pCM66 (Marx & Lidstrom, 2001). The resultant plasmid was introduced into the rpoH mutant by electroporation (Coppi et al, 2001).…”

Identification of an upstream regulatory sequence that mediates the transcription of mox genes in Methylobacterium extorquens AM1

“…In order to test this hypothesis, the mxaF A-tract sequence, AAGAAA, was inserted 17 bp upstream of the E. coli lac promoter in the promoter-probe vector pCM130. The lac promoter had been previously shown to be a weak promoter in M. extorquens AM1 (Marx & Lidstrom, 2001). The cloned tac promoter had an activity of 56-60 mU, while the construct containing the AAGAAA showed an increase to 128-180 mU.…”