Enhanced production of ectoine from methane using metabolically engineered Methylomicrobium alcaliphilum 20Z

Background Ectoine (1,3,4,5-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an attractive compatible solute because of its wide industrial applications. Previous studies on the microbial production of ectoine have focused on sugar fermentation. Alternatively, methane can be used as an inexpensive and abundant resource for ectoine production by using the halophilic methanotroph, Methylomicrobium alcaliphilum 20Z. However, there are some limitations, including the low production of ectoine from methane and the limited tools for the genetic manipulation of methanotrophs to facilitate their use as industrial strains. Results We constructed M. alcaliphilum 20ZDP with a high conjugation efficiency and stability of the episomal plasmid by the removal of its native plasmid. To improve the ectoine production in M. alcaliphilum 20Z from methane, the ectD (encoding ectoine hydroxylase) and ectR (transcription repressor of the ectABC-ask operon) were deleted to reduce the formation of by-products (such as hydroxyectoine) and induce ectoine production. When the double mutant was batch cultured with methane, ectoine production was enhanced 1.6-fold compared to that obtained with M. alcaliphilum 20ZDP (45.58 mg/L vs. 27.26 mg/L) without growth inhibition. Notably, a maximum titer of 142.32 mg/L was reached by the use of an optimized medium for ectoine production containing 6% NaCl and 0.05 μM of tungsten without hydroxyectoine production. This result demonstrates the highest ectoine production from methane to date. Conclusions Ectoine production was significantly enhanced by the disruption of the ectD and ectR genes in M. alcaliphilum 20Z under optimized conditions favoring ectoine accumulation. We demonstrated effective genetic engineering in a methanotrophic bacterium, with enhanced production of ectoine from methane as the sole carbon source. This study suggests a potentially transformational path to commercial sugar-based ectoine production. Graphical Abstract

Complete Genome and Plasmid Sequences of the Psychrotolerant Aureimonas Strain SA4125, Isolated from Antarctic Moss Vegetation

The complete genome sequences of Aureimonas sp. strain SA4125 and its native plasmid pSA4125 were determined. The genome sequence comprises 4,968,066 bp, with a GC content of 66.0%, and contains 4,691 coding DNA sequences (CDSs), 3 rRNA operons, and 50 tRNAs. The native plasmid comprises 131,777 bp, with a GC content of 62.3%, and contains 138 CDSs.

Enhanced Production of Ectoine From Methane Using Metabolically Engineered Methylomicrobium Alcaliphilum 20Z

Background: Ectoine (1,3,4,5-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an attractive compatible solute because of its wide industrial applications. Previous studies on the microbial production of ectoine have focused on sugar fermentation. Alternatively, methane can be used as an inexpensive and abundant resource for ectoine production by using the halophilic methanotroph, Methylomicrobium alcaliphilum 20Z. However, there are some limitations, including the low production of ectoine from methane and the limited tools for the genetic manipulation of methanotrophs to facilitate their use as industrial strains.Results: We constructed a genetically tractable M. alcaliphilum 20Z with a high conjugation efficiency and stability of the episomal plasmid by the removal of its native plasmid. To improve the ectoine production in M. alcaliphilum 20Z from methane, the ectD (encoding ectoine hydroxylase) and ectR (transcription repressor of the ectABC-ask operon) were deleted to reduce the formation of by-products (such as hydroxyectoine) and induce ectoine production. When the double mutant was batch cultured with methane, ectoine production was enhanced 1.6-fold compared to that obtained with M. alcaliphilum 20ZDP (45.58 mg/L vs. 27.26 mg/L) without growth inhibition. Notably, the use of an optimized medium for ectoine production, containing 6% NaCl and 0.05 µM tungsten, gave ectoine yields of up to 142.32 mg/L without hydroxyectoine production. This result demonstrates the highest ectoine production from methane to date.Conclusions: Ectoine production was significantly enhanced by the disruption of the ectD and ectR genes in M. alcaliphilum 20Z under optimized conditions favoring ectoine accumulation. We demonstrated effective genetic engineering in a methanotrophic bacterium, with enhanced production of ectoine from methane as the sole carbon source. This study suggests a potentially transformational path to commercial sugar-based ectoine production.

Non-intertwined strands of plasmid DNA contradict the Watson and Crick model of DNA structure

According to Watson and Crick (W/C) model of DNA structure, a DNA molecule consists of two antiparallel polynucleotide chains, intertwined with each other. Although W/C model is accepted widely, some researchers have raised questions against it and proposed alternative structures for DNA. In the present study, we examined W/C model using plasmid DNA. It was hypothesized that two strands of plasmid will remain intertwined (and not separate from each other) under denaturing conditions if it follows W/C model. To test this, plasmid DNA was denatured using sodium hydroxide (NaOH) and analyzed by agarose gel electrophoresis. It was observed that addition of 0.5 N NaOH to pUC19 or pBR322 plasmids resulted in a new form of DNA having higher electrophoretic mobility in agarose gel. Higher electrophoretic mobility DNA (HmDNA) in NaOH-denatured pUC19 was digestible with S1 nuclease, but not with HindIII and ‘exonuclease I + alkaline phosphatase’. These results demonstrated that HmDNA is single-stranded circular DNA, formed due to separation of two strands of NaOH-denatured plasmid. Single-stranded and circular nature of HmDNA was corroborated by its comparable electrophoretic mobility with purified top and bottom strands of plasmid DNA. Next, we examined whether HmDNA can re-anneal into the native plasmid. Interestingly, when subjected to renaturing conditions, HmDNA from NaOH-denatured pUC19 re-annealed to form native pUC19 plasmid, which was digestible with HindIII and induced ampicillin resistance in Escherichia coli. These findings demonstrated the reversible separation of two strands of plasmid DNA and contradicted the W/C model of DNA structure.

Non-intertwined strands of plasmid DNA contradicts the Watson and Crick model of DNA structure

According to Watson and Crick (W/C) model of DNA structure, a DNA molecule consists of two antiparallel polynucleotide chains, intertwined with each other. Although W/C model is accepted widely, some researchers have raised questions against it and proposed alternative structures for DNA. In the present study, we examined W/C model using plasmid DNA. It was hypothesized that two strands of plasmid will remain intertwined (and not separate from each other) under denaturing conditions if it follows W/C model. To test this, plasmid DNA was denatured using sodium hydroxide (NaOH) and analyzed by agarose gel electrophoresis. It was observed that addition of NaOH to pUC19 and pBR322 plasmids resulted in a new form of DNA having higher electrophoretic mobility in agarose gel. Higher electrophoretic mobility DNA (HmDNA) in NaOH-denatured pUC19 was digestible with S1 nuclease, but not with HindIII and ‘exonuclease I + alkaline phosphatase’. These results demonstrated that HmDNA is single-stranded circular DNA, formed due to separation of two strands of NaOH-denatured plasmid. Single-stranded and circular nature of HmDNA was corroborated by its comparable electrophoretic mobility with purified top and bottom strands of plasmid DNA. Next, we examined whether HmDNA can re-anneal into the native plasmid. Interestingly, when HmDNA from NaOH-denatured pUC19 was subjected to renaturing conditions, it formed native pUC19 plasmid, which was digestible with HindIII and induced ampicillin resistance in Escherichia coli cells. These findings demonstrated the reversible separation of two strands of plasmid DNA and contradicted the W/C model of DNA structure.

Methylome and Complete Genome Sequence of Parageobacillus toebii DSM 14590T, a Thermophilic Bacterium

ABSTRACT Here, we present the first complete genome assembly of the thermophilic bacterium Parageobacillus toebii DSM 14590T. The P. toebii DSM 14590T genome consists of a 3,270,071-bp circular chromosome and a 52,989-bp native plasmid.

Native Plasmid-Encoded Mercury Resistance Genes Are Functional and Demonstrate Natural Transformation in Environmental Bacterial Isolates

ABSTRACT Plasmid-mediated horizontal gene transfer (HGT) is a major driver of genetic diversity in bacteria. We experimentally validated the function of a putative mercury resistance operon present on an abundant 8-kbp native plasmid found in groundwater samples without detectable levels of mercury. Phylogenetic analyses of the plasmid-encoded mercury reductases from the studied groundwater site show them to be distinct from those reported in proximal metal-contaminated sites. We synthesized the entire native plasmid and demonstrated that the plasmid was sufficient to confer functional mercury resistance in Escherichia coli. Given the possibility that natural transformation is a prevalent HGT mechanism in the low-cell-density environments of groundwaters, we also assayed bacterial strains from this environment for competence. We used the native plasmid-encoded metal resistance to design a screen and identified 17 strains positive for natural transformation. We selected 2 of the positive strains along with a model bacterium to fully confirm HGT via natural transformation. From an ecological perspective, the role of the native plasmid population in providing advantageous traits combined with the microbiome’s capacity to take up environmental DNA enables rapid adaptation to environmental stresses. IMPORTANCE Horizontal transfer of mobile genetic elements via natural transformation has been poorly understood in environmental microbes. Here, we confirm the functionality of a native plasmid-encoded mercury resistance operon in a model microbe and then query for the dissemination of this resistance trait via natural transformation into environmental bacterial isolates. We identified 17 strains including Gram-positive and Gram-negative bacteria to be naturally competent. These strains were able to successfully take up the plasmid DNA and obtain a clear growth advantage in the presence of mercury. Our study provides important insights into gene dissemination via natural transformation enabling rapid adaptation to dynamic stresses in groundwater environments.

Native plasmid-encoded mercury resistance genes are functional and demonstrate natural transformation in environmental bacterial isolates

Plasmid-mediated horizontal gene transfer (HGT) is a major driver of genetic diversity in bacteria. We experimentally validated the function of a putative mercury resistance operon present on an abundant 8 kilobase pair native plasmid found in groundwater samples without detectable levels of mercury. Phylogenetic analyses of the plasmid-encoded mercury reductases from the studied groundwater site show them to be distinct from those reported in proximal metal-contaminated sites. We synthesized the entire native plasmid and demonstrated that the plasmid was sufficient to confer functional mercury resistance in Escherichia coli. Given the possibility that natural transformation is a prevalent HGT mechanism in the low cell density environments of groundwaters, we also assayed bacterial strains from this environment for competence. We used the native plasmid-encoded metal resistance to design a screen and identified 17 strains positive for natural transformation. We selected 2 of the positive strains along with a model bacterium to fully confirm HGT via natural transformation. From an ecological perspective, the role of the native plasmid population in providing advantageous traits combined with the microbiome’s capacity to take up environmental DNA enables rapid adaptation to environmental stresses.ImportanceHorizontal transfer of mobile genetic elements via natural transformation has been poorly understood in environmental microbes. Here, we confirm the functionality of a native plasmid-encoded mercury resistance operon in a model microbe and then query for the dissemination of this resistance trait via natural transformation into environmental bacterial isolates. We identify seventeen strains including Gram-positive and Gram-negative bacteria to be naturally competent. These strains were able to successfully take up the plasmid DNA and obtain a clear growth advantage in the presence of mercury. Our study provides important insights into gene dissemination via natural transformation enabling rapid adaptation to dynamic stresses in groundwater environments.

Draft Genome Sequences of Sporulation-Impaired Bacillus pumilus Strain NRS576 and Its Native Plasmid p576

Bacillus pumilus spores can cause foodborne poisonings. B. pumilus strain NRS576 forms spores with a very reduced efficiency due to the presence of a plasmid, named p576.