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Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

ABSTRACT: We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus.

SUBMITTER: Cue D

PROVIDER: S-EPMC168436 | biostudies-other | 1997 Apr

REPOSITORIES: biostudies-other

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Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

Cue D D Lam H H Dillingham R L RL Hanson R S RS Flickinger M C MC

Applied and environmental microbiology 19970401 4

We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two l ...[more]

PMID: 9097439

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Project description:Bacillus methanolicus MGA3 is a thermotolerant and relatively fast-growing methylotroph able to secrete large quantities of glutamate and lysine. These natural characteristics make B. methanolicus a good candidate to become a new industrial chassis organism, especially in a methanol-based economy. Intriguingly, the only substrates known to support B. methanolicus growth as sole sources of carbon and energy are methanol, mannitol, and, to a lesser extent, glucose and arabitol. Because fluxomics provides the most direct readout of the cellular phenotype, we hypothesized that comparing methylotrophic and nonmethylotrophic metabolic states at the flux level would yield new insights into MGA3 metabolism. In this study, we designed and performed a 13C metabolic flux analysis (13C-MFA) of the facultative methylotroph B. methanolicus MGA3 growing on methanol, mannitol, and arabitol to compare the associated metabolic states. On methanol, results showed a greater flux in the ribulose monophosphate (RuMP) pathway than in the tricarboxylic acid (TCA) cycle, thus validating previous findings on the methylotrophy of B. methanolicus New insights related to the utilization of cyclic RuMP versus linear dissimilation pathways and between the RuMP variants were generated. Importantly, we demonstrated that the linear detoxification pathways and the malic enzyme shared with the pentose phosphate pathway have an important role in cofactor regeneration. Finally, we identified, for the first time, the metabolic pathway used to assimilate arabitol. Overall, those data provide a better understanding of this strain under various environmental conditions.IMPORTANCE Methanol is inexpensive, is easy to transport, and can be produced both from renewable and from fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as Bacillus methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories or to design methylotrophic capability in other strains already used by the industry.

Project description:BACKGROUND:Bacillus cereus is a bacterial species which grows efficiently on a wide range of carbon sources and accumulates biopolymer poly-hydroxybutyrate (PHB) up to 80% cell dry weight. PHB is an aliphatic polymer produced and stored intracellularly as a reservoir of carbon and energy, its mobilization is a key biological process for sporulation in Bacillus spp. Previously, B. cereus tsu1 was isolated and cultured on rapeseed cake substrate (RCS), with maximum of PHB accumulation reached within 12?h, and depleted after 48?h. Fore-spore and spore structure were observed after 24?h culture. RESULTS:Quantitative proteomic analysis of B. cereus tsu1 identified 2952 quantifiable proteins, and 244 significantly changed proteins (SCPs) in the 24?h:12?h pair of samples, and 325 SCPs in the 48?h:12?h pair of samples. Based on gene ontology classification analysis, biological processes enriched only in the 24?h:12?h SCPs include purine nucleotide metabolism, protein folding, metal ion homeostasis, response to stress, carboxylic acid catabolism, and cellular amino acid catabolism. The 48?h:12?h SCPs were enriched into processes including carbohydrate metabolism, protein metabolism, oxidative phosphorylation, and formation of translation ternary structure. A key enzyme for PHB metabolism, poly(R)-hydroxyalkanoic acid synthase (PhaC, KGT44865) accumulated significantly higher in 12?h-culture. Sporulation related proteins SigF and SpoEII were significantly higher in 24?h-samples. Enzymes for nitrate respiration and fermentation accumulated to the highest abundance level in 48?h-culture. CONCLUSIONS:Changes in proteome of B. cereus tsu1 during PHB intracellular mobilization were characterized in this study. The key enzyme PhaC for PHB synthesis increased significantly after 12?h-culture which supports the highest PHB accumulation at this time point. The protein abundance level of SpoIIE and SigF also increased, correlating with sporulation in 24?h-culture. Enzymes for nitrate respiration and fermentation were significantly induced in 48?h-culture which indicates the depletion of oxygen at this stage and carbon flow towards fermentative growth. Results from this study provide insights into proteome profile changes during PHB accumulation and reuse, which can be applied to achieve a higher PHB yield and to improve bacterial growth performance and stress resistance.

Dataset Information

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

Publications

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

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