Nanobacteria
7 facts about the history of the discovery of ultramicrobacteria
and the minimum size of a living cell
Svetlana Dedysh, Post-science
Nanobacteria are dwarf bacteria ("nanos" in Latin means "dwarf"), whose sizes lie in the nanometer range.
If the sizes of the vast majority of bacteria well studied by microbiologists are in the range from one to tens and hundreds of micrometers (millionths of a meter), then the sizes of nanobacteria are calculated in nanometers (billionths of a meter). It is the size of these organisms that is the limitation that has held back the study of nanobacteria for a long time.
1. The size of nanobacteriaThe resolution limit of the instrument used in routine practice to study bacteria, an optical microscope, is 0.2 micrometers, or 200 nanometers.
Objects smaller than two hundred nanometers are not visible in an optical microscope. Since the optical microscope is the main working tool of most microbiological laboratories in the world, it becomes clear why nanobacteria have not been in the field of view of microbiologists for a long time. To detect nanobacteria, an electron microscope is needed, which is not available in every laboratory or institute.
The size of 0.2 micrometers, or 200 nanometers, corresponds to the diameter of the pores of filters that are currently used to filter samples of water, serums and various solutions in order to purify them from bacterial cells. It is generally believed that solutions passed through such filters no longer contain microbial cells. Nanobacteria, however, pass through these filters, so the second term used to refer to these organisms is the term "filtering bacteria". The third synonym term is "ultramicrobacteria". This term is the most correct from a scientific point of view and is the most widely used.
What is the numerical definition of the category "ultramicrobacteria"? This category currently includes microbial cells whose volume does not exceed one tenth of a cubic micrometer. Based on the formulas for determining the volumes of geometric shapes, these are either spherical cells with a diameter of no more than 500 nanometers, or very thin sticks with a diameter of 150-200 nanometers and a length of up to several micrometers. Such microbial cells fall into the category of ultramicrobacteria.
2. The history of the discovery of ultramicrobacteriaThe first reports of the formation of ultrafine cells by some bacteria date back to the beginning of the last century – Sherman, Safford:
The occurrence of filterable forms of bacteria in nature and Primitive or filterable forms of bacteria (Science, 1928). These observations were made through the use of filtration to sterilize media and solutions. As it turned out, filtration through filters used at that time with a pore size of 0.45 microns did not lead to the desired result of obtaining sterile media. Later, the filtering cells of microorganisms were detected not only in laboratory cultures, but also in natural environments. In particular, the study of water, soil and other samples from different ecosystems using electron microscopy showed that microbial cells of ultra-small sizes are ubiquitous.
Until 1998, however, all such studies were in the nature of natural science observations. And in 1998, the Journal of Reports of the American Academy of Sciences (PNAS) published the work of Kajander E.O. and Ciftcioglu N. Nanobacteria – an alternative mechanism for pathogenic intra- and extracellular calcification and stone formation, in which it was stated that nanoscale particles (from 20 to 500 nanometers in diameter) were detected in human blood serum, shaped like microbial cells. Ultrathin sections of these objects also showed some similarity with sections of bacterial cells. As it was shown, these nanoobjects acted as calcium phosphate deposition centers, which served as the basis for the assumption of their role in the processes of stone formation and vascular calcification. It was claimed that the identified objects are capable of slow reproduction and are resistant to heating. The data of their molecular identification by determining the sequence of the 16S rRNA gene were also given, according to which they belonged to the class Alphaproteobacteria and were phylogenetically close to the known pathogens of the genera Brucella and Bartonella. The identified objects were proposed to be attributed to a new genus – the genus Nanobacterium, from which the term "nanobacteria" originated.
This message aroused great interest not only among doctors, but also specialists from various neighboring areas. In the subsequent flurry of publications, nanobacteria were identified almost everywhere – both in living and inanimate objects. A particularly large number of reports on the detection of nanobacteria have been made by geologists. The conclusions of the latter were based only on the observation of ultra-shallow objects (100-300 nanometers or less in diameter) in various geological rocks. In the overwhelming majority of such publications, no evidence was obtained in favor of the biological nature of the observed objects.
3. Minimum dimensions of a living cellThe growing number of speculative publications on the widespread detection of nanobacteria has forced microbiologists to seriously think about how small a microbial cell can be.
For this purpose, an emergency workshop was convened in 1999, at which the issue of the minimum size of microorganisms was discussed (Size Limits of Very Small Microorganisms, Proceedings of a Workshop, Washington DC, 1999).
The consensus accepted by the majority of scientists regarding the minimum size that ensures the independent existence of a living cell is reduced to a volume of 0.014 to 0.06 cubic micrometers and a diameter of 150 to 300 nanometers. These calculations are based on the fact that a growing and dividing cell must have a certain minimum volume to accommodate the "metabolic machine" that ensures its functioning. Of course, the necessary components are DNA, RNA, ribosomes, transcription and translation enzymes, a minimum set of proteins and lipids, cytosol substances and the corresponding periplasmic space. Thus, if the size of one ribosome is 15-20 nanometers, then the entire cell, which must contain at least a hundred ribosomes, cannot possibly have a diameter of 20 nanometers.
Figure from the article Nanobacteria revelations provoke new controversy (New Scientist, 2004) – VM.
4. About nanobacteria in blood serum10 years after the publication of the report on the detection of nanobacteria in blood serum, another study was published in the same journal (PNAS) – Marte, Young (2008).
Purported nanobacteria in human blood as calcium carbonate nanoparticles. It elegantly proved that particles completely identical to nanobacteria in appearance can be formed in blood serum as a result of chemical precipitation of calcium carbonate, and these microparticles, in turn, serve as centers of precipitation of calcium phosphate. In this work, a number of exhaustive proofs of the non-biological nature of the nanoparticles observed in blood serum were also given. In addition, nanobacteria from the blood could not be isolated in cultures. The data of their molecular identification with the accumulation of an extensive database of 16S rRNA gene sequences began to look quite different. The sequences of nanobacteria identified in the 1998 work turned out to be identical to those of representatives of the genus Phyllobacterium, widespread non-pathogenic heterotrophs, which are common pollutants (contaminants) of laboratory utensils and media. Thus, the existence of specific bacteria of the genus Nanobacterium has been refuted, which, however, does not refute the existence of microorganisms with nanoscale cells.
5. Proof of the existence of nanobacteriaDespite the abundance of contradictory information, as well as a number of outright speculations regarding nanobacteria, microbial cells of the nanoscale range exist.
To date, microbiologists have known a number of bacteria and archaea that have cells with a volume of less than 0.1 cubic micrometer. Many of these microorganisms are even obtained in pure cultures and are well studied. A characteristic feature of such microorganisms is the small size of the genome – from 0.5 to 3.2 million base pairs.
One example of ultramicrobacteria widespread in oceanic waters is Pelagibacter ubique. According to molecular data, up to 20% of all nucleotide sequences of 16S rRNA genes detected in ocean water belong to bacteria of this genus and species – Giovannoni et al., Genome streaming in a cosmopolitan oceanic bacterium (Science, 2005). It is really a very small bacterium that passes through the pores of bacterial filters, and the size of its genome is also very small – 1.3 million base pairs. It is an absolutely free-living, non-parasitic bacterium that grows very slowly. There are many examples of such organisms. In nature, such forms are not uncommon at all.
6. Dangerous ultramicrobacteriaAre there any potentially dangerous ultramicrobacteria?
Yes, indeed, there are such examples. One such example is Minibacterium massiliensis, a motile bacterium that has been isolated from the so–called ultrapure water used in hospitals for hemodialysis. It turned out that such bacteria are able to go through several stages of purification, including not only filtration, but also UV treatment. These bacteria belong to the class Betaproteobacteria. The genome of Minibacterium massiliensis was not so small – about 4 million base pairs – Audic et al., Genome analysis of Minibacterium massiliensis highlights the convergent evolution of water-living bacteria (PLoS Genet, 2007).
When analyzing the genome, multiple factors of resistance to antibiotics and heavy metals were found, as well as more than one hundred and fifty virulence factors. Other examples of pathogenic bacteria with nanoscale cells are well-studied microorganisms such as Brucella, Mycoplasma, Rickettsia and Chlamydia.
7. Ultramicrobacteria in natural drinking waterIn recent decades, the question of the presence of ultramicrobacteria in drinking natural water has been widely investigated.
These studies were initiated by Swiss microbiologists, who took into account the filtering cells not only in the reservoirs of Switzerland, but also in the famous Swiss bottled water. It turned out that even in bottled water, the number of cells of such microorganisms is thousands of cells in one milliliter. There are an order of magnitude more of them in natural water – tens of thousands of cells in one milliliter.
Later, attempts were made to isolate these organisms in pure cultures. In particular, a lot of work on the isolation of such cells has been carried out by Austrian microbiologists, but we have also studied filtering microorganisms in recent years. In particular, our laboratory conducted a study of the waters of natural reservoirs in the catchments of the Upper Volga and the waters of the Rybinsk reservoir. Our studies have shown that the number of filtered microorganisms in them is quite high, amounting to tens of thousands of cells in one milliliter of water – Fedotov et al., Filtered forms of microorganisms in the waters of the Rybinsk reservoir (Microbiology, 2013). Among these cells there are both bacteria and archaea of completely unknown groups. The study of the genomic potential and, accordingly, the functional potential of these organisms is an important task for further research.
About the author:
Svetlana Dedysh – Doctor of Biological Sciences, Head of the Laboratory of Microbiology of Swamp Ecosystems INMI RAS.
Portal "Eternal youth" http://vechnayamolodost.ru28.08.2014