Genetic manipulation of the restricted facultative methylotroph Hyphomicrobium X by the R-plasmid-mediated introduction of the Escherichia coli pdh genes (original) (raw)
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Applied Microbiology and Biotechnology, 2008
A phage Mu-driven two-plasmid system for DNA integration in Escherichia coli genome has been adjusted for Methylophilus methylotrophus. Constructed helper plasmids with broad-host-range replicons carry thermo-inducible genes for transposition factors MuA and MuB. Integrative plasmids that are only replicated in E. coli could be mobilized to M. methylotrophus and contained mini-Mu unit with a short terminus of Mu DNA, Mu-attL/ R. Mini-Mu unit was integrated in the M. methylotrophus genome via mobilization of the integrative plasmid to the cells carrying the helper in conditions of thermo-induced expression of MuA and MuB. In this system, mini-Mu unit was mainly integrated due to replicative transposition, and the integrated copy could be amplified in the M. methylotrophus chromosome in the presence of helper plasmid. A kan-gene flanked by FRT sites was inserted in one of the mini-Mu units, and it could be readily excised by yeast FLP recombinase that is encoded by the designed plasmid. The multiple Mu-driven gene insertion was carried out by integration of the Bacillus amyloliquefaciens α-amylase gene followed by curing the Km R marker before integration of the second mini-Mu unit with Pseudomonas putida xylE gene encoding catechol 2,3-dioxygenase (C23O).
Genetic Tools for the Industrially Promising Methanotroph Methylomicrobium buryatense
Applied and Environmental Microbiology, 2014
Aerobic methanotrophs oxidize methane at ambient temperatures and pressures and are therefore attractive systems for methane-based bioconversions. In this work, we developed and validated genetic tools for Methylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph. M. buryatense was isolated directly on natural gas and grows robustly in pure culture with a 3-h doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept, we used a sucrose counterselection system to eliminate glycogen production in M. buryatense by constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractable variant strain that can be conjugated with small incompatibility group P (IncP)-based broad-host-range vectors and determined that this capability is due to loss of the native plasmid. These tools make M. buryatense a promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane.
Chloromethane-Dependent Expression of the cmu Gene Cluster of Hyphomicrobium chloromethanicum
Applied and Environmental Microbiology, 2004
The methylotrophic bacterium Hyphomicrobium chloromethanicum CM2 can utilize chloromethane (CH(3)Cl) as the sole carbon and energy source. Previously genes cmuB, cmuC, cmuA, and folD were shown to be essential for the growth of Methylobacterium chloromethanicum on CH(3)Cl. These CH(3)Cl-specific genes were subsequently detected in H. chloromethanicum. Transposon and marker exchange mutagenesis studies were carried out to identify the genes essential for CH(3)Cl metabolism in H. chloromethanicum. New developments in genetic manipulation of Hyphomicrobium are presented in this study. An electroporation protocol has been optimized and successfully applied for transformation of mutagenesis plasmids into H. chloromethanicum to generate stable CH(3)Cl-negative mutants. Both transposon and marker exchange mutageneses were highly applicable for genetic analysis of Hyphomicrobium. A reliable and reproducible selection procedure for screening of CH(3)Cl utilization-negative mutants has also been developed. Mutational inactivation of cmuB, cmuC, or hutI resulted in strains that were unable to utilize CH(3)Cl or to express the CH(3)Cl-dependent polypeptide CmuA. Reverse transcription-PCR analysis indicated that cmuB, cmuC, cmuA, fmdB, paaE, hutI, and metF formed a single cmuBCA-metF operon and were coregulated and coexpressed in H. chloromethanicum. This finding led to the conclusion that, in cmuB and cmuC mutants, impaired expression of cmuA was likely to be due to a polar effect of the defective gene (cmuB or cmuC) located upstream (5') of cmuA. The detrimental effect of mutation in hutI on the upstream (5')-located cmuA is not clear but indicated that all the genes located within the cmuBCA-metF operon are coordinately expressed. Expression of the cmuBCA-metF transcript was also shown to be strictly CH(3)Cl inducible and was not repressed by the alternative C(1) substrate methanol. Sequence analysis of a transposon mutant (D20) led to the discovery of the previously undetected hutI and metF genes located 3' of the paaE gene in H. chloromethanicum. MetF, a putative methylene-tetrahydrofolate reductase, had 27% identity to MetF from M. chloromethanicum. Mutational and transcriptional analysis data indicated that, in H. chloromethanicum, CH(3)Cl is metabolized via a corrinoid-specific (cmuA) and tetrahydrofolate-dependent (metF, purU, folD) methyltransfer system.
Journal of Biological Chemistry
A 1476-base pair DNA fragment from Haemophilus haemolyticus containing the HhaI methyltransferase gene was isolated from a cell library and cloned into pBR322. The nucleotide sequence of this fragment was determined. The structural gene is 981 nucleotides in length coding for a protein of 327 amino acids (M, 37,000). The translational start signal (ATG) is preceded by the putative ribosome-binding site (TAAG). Recombinant plasmids containing the 1476-basepair fragment are completely methylated when isolated from Escherichia coli, as judged by their insusceptibility to the HhaI restriction endonuclease. However, the presence of an active HhaI methylase gene in certain E. coli strains results in a very poor yield of transformants and/or in vivo-originated deletions due to the Rgl functions of these hosts. The in vivo transcription initiation sites have been identified by S1 protection and primer-extension experiments using specific probes with total RNA prepared from E. coli cells (HB101 or RR1) which tolerate the expression of MHhaI.
Molecular Toolbox for Genetic Manipulation of the Stalked Budding Bacterium Hyphomonas neptunium
Applied and Environmental Microbiology, 2014
The alphaproteobacterium Hyphomonas neptunium proliferates by a unique budding mechanism in which daughter cells emerge from the end of a stalk-like extension emanating from the mother cell body. Studies of this species so far have been hampered by the lack of a genetic system and of molecular tools allowing the regulated expression of target genes. Based on microarray analyses, this work identifies two H. neptunium promoters that are activated specifically by copper and zinc. Functional analyses show that they have low basal activity and a high dynamic range, meeting the requirements for use as a multipurpose expression system. To facilitate their application, the two promoters were incorporated into a set of integrative plasmids, featuring a choice of two different selection markers and various fluorescent protein genes. These constructs enable the straightforward generation and heavy metal-inducible synthesis of fluorescent protein fusions in H. neptunium, thereby opening the door to an in-depth analysis of polar growth and development in this species. Citation Jung A, Eisheuer S, Cserti E, Leicht O, Strobel W, Möll A, Schlimpert S, Kühn J, Thanbichler M. 2015. Molecular toolbox for genetic manipulation of the stalked budding bacterium Hyphomonas neptunium. Appl Environ Microbiol 81:736 -744.
Transfer of broad host range plasmids to the type I obligate methanotroph Methylomonas albus
FEMS Microbiology Letters, 1987
Plasmids RP4 (IncP) and R300B (IncQ) (mobilized by RP4) were transferred to Methylomonas albus from Escherichia coli donors at frequencies of 2 × 10 3 and 1-10 3 per donor. BY contrast, plasmids pS-a (IncW) and the IncP plasmids R68.45, R751 and derivatives carrying bacteriophage Mu and/or transposons were transferred to M. albus from E. coli at frequencies of 10 7-10-8 per donor. M. albus transconjugants carrying R68.45, R751 and their derivatives were isolated and used as donors in matings with either other strains of M. albus or E. coli. Plasmids carrying bacteriophage Mu were transferred to E. coli at a frequency of 10 5-10-6 per donor and 10 2-10 3 per donor for those not carrying Mu. Transfer to M. albus recipients was at a frequency of 10 3-10 -4 per donor regardless of the presence or absence of Mu, indicating the absence of a suicide effect. Plasmids carrying Tn5 were found in M. albus to determine simultaneous kanamycin (Km) and streptomycin (Sm) resistance.
FEMS Microbiology Letters, 2006
Many strcptomycetes, including S. ~or/i~o/o~ A3(2). possess a potent methyl-specific restriction system which can present an effcctivc barrier to the introduction of hcterologous DNA. We have compared the efficiency of intergeneric conjugal transfer of different types of plasmids to S. ~oc/i~o/or and S. /i~~rt/trr~.r 66 using two C: c~oli donors: the standard, methylation proficient strain Sl7-I. and the methylation deficient donor. E:Tl2567(pUB307). We demonstrate that the mcthylation delicient donor can yield > IO'-fold more S'. CY~C~/~C~O/O~~ exconjugant5 than the standard donor. In the case of pSETl52 derivatives, which integrate into the host chromosome by site-specific recombination. up to IO% o f streptomycete spores in the conjugation mixture inherit the plasmid. The conjugation procedure is cfticient enough to obtain exconjugants with 'suicide' delivery plasmids and thercforc provides a simple route for conducting gene disruptions in methyl DNA-restricting streptomycetes. and possibly other bacteria.