Bacteria are generally assumed to be monoploid (haploid). This assumption is mainly based on generalization of the results obtained with the most intensely studied model bacterium, Escherichia coli (a gamma-proteobacterium), which is monoploid during very slow growth. However, several species of proteobacteria are oligo- or polyploid, respectively. To get a better overview of the distribution of ploidy levels, genome copy numbers were quantified in four species of three different groups of proteobacteria. A recently developed Real Time PCR approach, which had been used to determine the ploidy levels of halophilic archaea, was optimized for the quantification of genome copy numbers of bacteria. Slow-growing (doubling time 103 minutes) and fast-growing (doubling time 25 minutes) E. coli cultures were used as a positive control. The copy numbers of the origin and terminus region of the chromosome were determined and the results were in excellent agreement with published data. The approach was also used to determine the ploidy levels of Caulobacter crescentus (an alpha-proteobacterium) and Wolinella succinogenes (an epsilon-proteobacterium), both of which are monoploid. In contrast, Pseudomonas putida (a gamma-proteobacterium) contains 20 genome copies and is thus polyploid. A survey of the proteobacteria with experimentally-determined genome copy numbers revealed that only three to four of 11 species are monoploid and thus monoploidy is not typical for proteobacteria. The ploidy level is not conserved within the groups of proteobacteria, and there are no obvious correlations between the ploidy levels with other parameters like genome size, optimal growth temperature or mode of life.

/pdf/quantification-of-ploidy-in-proteobacteria-revealed-the-2d1p8dgz61.pdf

Quantification of Ploidy in Proteobacteria Revealed the Existence of Monoploid, (Mero-)Oligoploid and Polyploid Species

During recent years, it has become clear that many species of archaea and bacteria are polyploid and contain more than 10 copies of their chromosome. In this contribution, eight examples are discussed to highlight different aspects of polyploidy in prokaryotes. The species discussed are the bacteria Azotobacter vinelandii, Deinococcus radiodurans, Sinorhizobium meliloti, and Epulopiscium as well as the archaea Methanocaldococcus jannaschii, Methanococcus maripaludis, Haloferax volcanii, and haloarchaeal isolates from salt deposits. The topics include possible laboratory artifacts, resistance against double-strand breaks, long-term survival, relaxation of DNA segregation and septum formation, enforced polyploidy by a eukaryotic host, genome equalization by gene conversion, and the nongenetic usage of genomic DNA as a phosphate storage polymer. Together, the selected topics give an overview of the biodiversity of polyploidy in archaea and bacteria.

Polyploidy in archaea and bacteria: about desiccation resistance, giant cell size, long-term survival, enforcement by a eukaryotic host and additional aspects.

The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world’s increasing demand for sustainable food, fibres and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalysed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array (n=16) of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B,D,E,F,H,J,K,M,N,Q,S,U,V,X,Y and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B,S,H,Y) were successfully expressed, demonstrating that multiple components of nitrogenase can targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.

/pdf/expression-of-16-nitrogenase-proteins-within-the-plant-4byh9eweu3.pdf

Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix.

Re-engineering of complex biological systems (CBS) is an important goal for applications in synthetic biology. Efforts have been made to simplify CBS by refactoring a large number of genes with rearranged polycistrons and synthetic regulatory circuits. Here, a posttranslational protein-splicing strategy derived from RNA viruses was exploited to minimize gene numbers of the classic nitrogenase system, where the expression stoichiometry is particularly important. Operon-based nif genes from Klebsiella oxytoca were regrouped into giant genes either by fusing genes together or by expressing polyproteins that are subsequently cleaved with Tobacco Etch Virus protease. After several rounds of selection based on protein expression levels and tolerance toward a remnant C-terminal ENLYFQ-tail, a system with only five giant genes showed optimal nitrogenase activity and supported diazotrophic growth of Escherichia coli. This study provides an approach for efficient translation from an operon-based system into a polyprotein-based assembly that has the potential for portable and stoichiometric expression of the complex nitrogenase system in eukaryotic organisms.

https://www.pnas.org/content/pnas/115/36/E8509.full.pdf

Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology.

Summary


Haloferax volcanii is highly polyploid and contains about 20 copies of the major chromosome. A heterozygous strain was constructed that contained two different types of genomes: the leuB locus contained either the wild-type leuB gene or a leuB:trpA gene introduced by gene replacement. As the trpA locus is devoid of the wild-type trpA gene, growth in the absence of both amino acids is only possible when both types of genomes are simultaneously present, exemplifying gene redundancy and the potential to form heterozygous cells as one possible evolutionary advantage of polyploidy. The heterozygous strain was grown (i) in the presence of tryptophan, selecting for the presence of leuB, (ii) in the presence of leucine selecting for leuB:trpA and (iii) in the absence of selection. Both types of genomes were quantified with real-time PCR. The first condition led to a complete loss of leuB:trpA-containing genomes, while under the second condition leuB-containing genomes were lost. Also in the absence of selection gene conversion led to a fast equalization of genomes and resulted in homozygous leuB-containing cells. Gene conversion leading to genome equalization can explain the escape from ‘Muller's ratchet’ as well as the ease of mutant construction using polyploid haloarchaea.

Gene conversion results in the equalization of genome copies in the polyploid haloarchaeon Haloferax volcanii

Functional expression of a fusion-dimeric MoFe protein of nitrogenase in Azotobacter vinelandii.

Several lines of experimental analyses on the ploidy status of Azotobacter vinelandii genome lead to the conclusion that it contains more than 40 copies of its chromosome and therefore it is a polyploid organism. The genetic evidence argues against the existence of polyploidy in these cells since the segregation pattern of genetic markers under lack of selection pressure mimic that of haploids. However, when A. vinelandii was made Nif− by inserting a kanamycin resistance marker gene in the nifDK sequence and the cells were selected for kanamycin resistance and Nif+ phenotype, we were able to score colonies that are both kanamycin resistant and Nif+. Therefore, when the cells were subjected to forced double selection of the same locus, they behaved as if they carried at least two chromosomes, one carrying the kanamycin resistance marker in the nifDK genes and the other carrying the intact nifDK genes. These analyses suggested that at least a diploidy status can be induced in these cells under selection pressure.

Isolation and characterization of nif::kanamycin and nitrogen fixation proficient Azotobacter vinelandii strain, and its implication on the status of multiple chromosomes in Azotobacter

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2800%2901847-0

Identification of a second site compensatory mutation in the Fe-protein that allows diazotrophic growth of Azotobacter vinelandii UW97.

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Functional expression of a fusion-dimeric MoFe protein of nitrogenase in Azotobacter vinelandii.

Isolation and characterization of nif::kanamycin and nitrogen fixation proficient Azotobacter vinelandii strain, and its implication on the status of multiple chromosomes in Azotobacter

Identification of a second site compensatory mutation in the Fe-protein that allows diazotrophic growth of Azotobacter vinelandii UW97.