Bacteria

Some General Characteristics

Bacteria are the most abundant and widespread cellular life forms on Earth. They are generally microscopic single-celled organisms that in some cases form relatively simple multicellular entities (Figure 1). Almost all bacteria have a cell wall. The characteristic component of that cell wall is a polymer called peptidoglycan. General characteristics of bacteria are described below.

Habitats

Bacteria have amassed an extraordinary number of adaptations and metabolic abilities, which have enabled them to survive in practically every habitat on earth. They are found in a diversity of aquatic and terrestrial environments— in waters, soils, rocks, and in the cells and/or bodies of other organisms.

They also occur in extreme environments such as high in the atmosphere (Smith et al. 2018); in the deepest oceanic trenches (Glud et al. 2013); in the deepest layer of ocean crust (Mason et al. 2010); in the driest deserts (Schulze-Makuch et al. 2018); in water bodies that are very acidic (Quatrini and Johnson 2018) or alkaline (Padan et al. 2005), and at extreme temperatures. Some bacteria remain biologically active at temperatures as low as -20°C, which is believed to be the lowest temperature limit for sustaining life (D'Amico et al. 2006, Rivkina et al. 2000); other bacteria are capable of growing at temperatures as high as 100°C (Kashefi et al. 2002).

Although some bacteria are able to survive long periods under very dry conditions, their cells ultimately require water for metabolic activity— this is true for all cells regardless of the type of organism. Thus, all bacterial cells (and all cells in general) are in essence aquatic. The so-called "terrestrial" bacteria actually live within thin films of water found on the surfaces of soils, plants or other objects, or within water-filled pores of rocks or soils (Fenchel 1994).

Cell Size

Bacteria typically have cell volumes ranging from around 0.4 to 3 µm³ (Levin and Angert 2015) and cell diameters that are almost always larger than 0.2 µm—which is the filter pore size used for "sterile" filtration. However, some bacteria are smaller, and others much larger, than those typical sizes. Within a given species, cell size is known to vary greatly with nutrient availability, with starved cells tending to be much smaller than cells growing under nutrient-replete conditions (Chien et al. 2013).

The smallest described free-living bacterial cells include a group of marine Actinobacteria with an average cell volume of approximately 0.013 µm³ (Ghai et al. 2013), and diverse bacterial taxa in groundwater with a median cell volume of 0.009±0.002 µm³ (Luef et al. 2015). At the other extreme are bacterial cells that are large enough to be seen with the naked eye, such as Thiomargarita namibiensis, which is spherical and has a cell diameter of 750 µm (Schulz et al. 1999), and Epulopiscium fishelsoni, with a cell size of approximately 80 by 600 µm (Angert et al. 1993). The largest known bacterium has not yet been grown in culture. It is Candidatus Thiomargarita magnifica and has an average cell length greater than 9,000 µm (Volland et al. 2022).

Cell Shape

Bacterial cells come in many different shapes (Figure 2). Many bacteria have scientific names that describe their shapes. Some cell shapes include:

• spheres— also known as cocci (singular: coccus), e.g., Streptococcus pyogenes, Staphylococcus aureus, and Veillonella parvula.

• rods—also referred to as bacilli (singular: bacillus), e.g., Lactobacillus acidophilus, Bacillus subtilis, and Clostridium botulinum.

• short rods or ovals— known as coccobacilli (singular: coccobacillus), e.g., Bordetella pertussis, Haemophilus influenzae, and Pasteurella multocida

• comma shaped (curved rods or incomplete spirals)— called vibrios, e.g., Vibrio cholerae, Butyrivibrio fibrisolvens, and Desulfovibrio desulfuricans.

• rigid spirals— called spirillas, e.g., Rhodospirillum rubrum, Helicobacter pylori, and Campylobacter jejuni.

• flexible spirals— called spirochaetes, e.g., Treponema pallidum, Borreliella (Borrelia) burgdorferi, and Leptospira interrogans.

• filamentous— with long narrow filament-like structures (modified cells) called hyphae. The hyphae grow by extension of the tip and by formation of new branches in a manner similar to the hyphae of fungi. Like fungi, some filamentous bacteria may form masses of branching hyphae known as mycelia. The Phylum Actinobacteriota (Actinobacteria) contains taxa that form hyphae as part of their normal life cycle, e.g., Streptomyces spp., Actinomadura spp., and Microbispora spp.

• prosthecate— having stalk-like cellular appendages called prosthecae (singular: prostheca). These appendages are narrowed portions of the cell. Like other parts of the cell they contain cytoplasm and are surrounded by cell membrane and cell wall. Some bacteria have a holdfast — gel-like adhesive material— at the end of their prostheca, which allows them to attach to surfaces. Examples of prosthecate bacteria include Hyphomonas neptunium, Caulobacter crescentus, and Asticcacaulis excentricus.

• variable shaped (with no characteristic shape)— also known as pleomorphic. e.g., Mycoplasma pneumoniae, Phytoplasma mali, and L-forms of Mycobacterium tuberculosis.

Cell Components and Structure

The cell of a bacterium has two main parts: (1) the cytoplasm and (2) the various layers that surround the cytoplasm, known as the cell envelope.

Cytoplasm

The bulk of a bacterial cell consists of cytoplasm. It includes the watery matrix of the cell, called the cytosol (sometimes referred to as the cytoplasmic matrix), and all cellular components found within the cytosol. These components include ribosomes— tiny complex structures that carry out protein synthesis; and the nucleoid— the region within the cell where the bacterial chromosome (or chromosomes) are located. Small extrachromosomal DNA molecules may also be found within the cytosol. In addition, bacterial cells may contain various inclusions (also called inclusion bodies)— materials stored directly within the cytosol or within special structures that are bounded by protein or lipid membrane. Examples of inclusions include:

• cyanophycin granules— these are inclusions that apparently lack any kind of surrounding membrane or proteinaceous shell (Allen and Weathers 1980, Seufferheld et al. 2003). They consist of a nitrogen-rich polymer called multi-L-arginyl poly-(L-aspartic acid), which contains equimolar amounts of arginine and aspartate (Allen and Weathers 1980, Klemke et al. 2016). Cyanophycin granules are found in many cyanobacteria and in some heterotrophic bacteria, and they apparently serve as a storage reserve for nitrogen, carbon, and energy (Füser and Steinbüchel, 2007).

• protein-bounded gas vesicles, which enable some bacteria to control their buoyancy and position within a water column. These vesicles are impermeable to water but highly permeable to gases (Walsby 1994). Gas vesicles are found mostly in planktonic bacteria and archaea, including many cyanobacteria, purple sulfur bacteria, green sulfur bacteria, and green nonsulfur bacteria (Walsby 1994);

• carboxysomes— protein-bounded structures found in cyanobacteria, many chemoautotrophs, and a few purple sulfur bacteria (Turmo et al. 2017). These structures contain concentrated CO₂ and enzymes needed for "fixing" CO₂ — i.e., for converting CO₂ into organic compounds (Turmo et al. 2017, Rae et al. 2013).

• lipid droplets— which contain lipids and are surrounded by a lipid membrane. The membrane consists of a monolayer of phospholipids with many proteins (Yang et al. 2012). Lipid droplets are found in the cells of bacteria, archaea, and eukaryotes— including humans (Murphy 2012), and may have been present in an ancient ancestor of these groups (Zhang and Liu 2017). They were originally believed to function only as storage reservoirs for lipids; however, they are now recognized as dynamic organelles with multiple functions, including the storage, transport, and metabolism of lipids, and the binding and protection of nucleic acids (Zhang and Liu 2017, Yang et al. 2012). Additional functions have been identified in eukaryotes, but have not yet been well studied in bacteria (Zhang and Liu 2017). Lipid droplets are known to occur in most bacteria (Docampo 2016).

• magnetosomes— organelles that consist of a magnetic iron-containing crystal, usually magnetite (Fe₃O₄) or greigite (Fe₃S₄), surrounded by a lipid bilayer membrane that contains proteins (Docampo 2016). These organelles are typically arranged in one or more chains within a cell. Bacteria containing magnetosomes— the so-called magnetotactic bacteria— use these organelles to align themselves with and swim along geomagnetic field lines (Yan et al. 2012). Magnetotactic bacteria have a gram-negative cell envelope, they are found in environments with low concentrations of molecular oxygen (i.e., they are typically anaerobes or microaerophiles), and they belong to at least six different phyla of bacteria— Desulfobacterota, Proteobacteria, Nitrospirota, and Planctomycetota; and the uncultured (candidate) phyla Omnitrophota (Omnitrophica) and Latescibacterota (Latescibacteria) (Lin et al. 2018, Yan et al. 2012, Genome Taxonomy Database 2018). The magnetosome genes in these different lineages appear to have a common origin (Lin et al. 2018).

• acidocalcisomes— acidic membrane-bound organelles containing phosphorous compounds (including polyphosphate, pyrophosphate, and orthophosphate) that are complexed with calcium and other cations (Docampo et al. 2010, Docampo 2016). These organelles also contain enzymes for the synthesis and degradation of pyrophosphate and polyphosphate (Docampo and Moreno 2011). The surrounding membrane consists of a lipid bilayer and contains proteins, including proton pumps, which maintain the acidity of the organelle (Docampo 2016). Acidocalcisomes are found in bacteria, eukaryotes (including humans), and probably archaea (Seufferheld et al 2011). They were previously referred to as volutin granules, metachromatic granules, or polyphosphate granules, vacuoles or bodies (Docampo et al. 2005, 2010; Seufferheld et al. 2011). Given their presence in the different cellular domains, these organelles, like lipid droplets, may have been present in an ancient ancestor, or they could be examples of convergent evolution (Seufferheld et al. 2011, Docampo 2016). Acidocalcisomes have been identified in the bacteria Agrobacterium tumefaciens and Rhodospirillum rubrum (Docampo 2016). They are important sites for the storage of cations and phosphorus (Docampo and Moreno 2011, Docampo et al. 2010). In eukaryotes, they are also known to be involved in calcium/cation homeostasis, regulation of intracellular pH, and osmoregulation (Docampo et al. 2010).

• anammoxosomes— membrane-bound organelles found in anaerobic ammonium-oxiding (anammox) bacteria from the phylum Planctomycetes (Order Brocadiales). The membrane surrounding this organelle consists of a lipid bilayer (Neumann et al. 2014). This bilayer is made up of unusual lipids called ladderanes and also contain proteins (Docampo 2016), including ATP synthase (de Almeida et al. 2015)— a molecular machine used to produce adenosine triphosphate (ATP). The anammoxosome is a dedicated compartment where the ammonium-oxidizing catabolic reactions take place and where usable energy— in the form of ATP— is produced (de Almeida et al. 2015). In the anammox reactions, ammonium (NH₄⁺) and nitrite (NO₂^-) are converted to nitrogen gas (N₂). In the process high energy electrons are captured, and fed into an electron transport chain. Energy released from the chemical reactions of the electron transport chain is used to drive proton pumps in the anammoxosome membrane, resulting in a highly energized membrane (due to a difference in proton concentration and charge across the membrane). The energy stored in the membrane is used to produce ATP. Thus the anammoxome, like the mitochondrion in eukaryotes, is believed to be an energy-generating organelle (Neumann et al. 2014, van Niftrik and Jetten 2012). Unlike the mitochondrion, the anammoxosome is believed to have originated from an invagination of the cytoplasmic membrane— because all membranes within the cell resemble one another in structure and lipid composition (Neumann et al. 2014). The anammoxome organelle has been successfully isolated from cells of anammox bacteria; thus it appears likely that the membrane surrounding this organelle is completely separate from the other membranes in the cell (Neumann et al. 2014).

Another feature that may be found in the cytoplasm of bacteria— at least within certain groups of bacteria— is intracytoplasmic membrane. Intracytoplasmic membranes are essentially invaginations of the cytoplasmic membrane. They can take the form of tubules, interconnected vesicles, and plate-like layers of membrane referred to as lamellae (Niederman 2006). These membranes may provide additional surface area for the production of ATP and they create compartments where metabolic processes can take place (Niederman 2006, LaSarre et al. 2018). The thylakoid membranes of cyanobacteria are examples of intracytoplasmic membrane. Bacteria that typically contain intracytoplasmic membranes include:

• phototrophic bacteria— which harvest energy from sunlight; examples include the cyanobacterium Synechococcus and the purple nonsulfur bacterium Rhodobacter;
• nitrifying bacteria— autotrophic bacteria that oxidize ammonia or nitrite to obtain energy; examples include the ammonia-oxidizing Nitrosomonas and the nitrite-oxidizing Nitrobacter.
• methane-utilizing bacteria— which generate energy by oxidizing methane; for example, Methylomicrobium album.
• bacteria from the phyla Planctomycetota (Planctomycetes) and Verrucomicrobiota (Verrucomicrobia)— these bacteria tend to have cells that are compartmentalized by internal membranes. For example, Gemmata obscuriglobus.

Note: there is some overlap when describing intracytoplasmic membrane and some of the membrane-bound organelles. For example, the membranes of magnetosomes have been found to be invaginations of the cytoplasmic membrane (Komeili et al. 2006) and thus could be described as intracytoplasmic membrane. Often it can be difficult to tell whether a membrane surrounding an internal compartment is completely separate from the cytoplasmic membrane.

Some bacteria may form intracytoplasmic membranes only under certain growth conditions. For example, in one study, cells of Methylobacterium organophilum produced intracytoplasmic membranes when they were grown in medium containing methane, but they failed to form intracytoplasmic membranes when they were given methanol or glucose as their sole carbon and energy source (Patt and Hanson 1978). Environmental conditions may also affect the amount of intracytoplasmic membrane that a cell produces. Photosynthetic bacteria may produce a greater amount of intracytoplasmic membrane under low light conditions than under high light conditions (Adamas and Hunter 2012). This is because under low light, there are fewer photons available per unit area and time, thus cells need to produce more pigments to effectively harvest light—and more intracytoplasmic membrane is necessary to contain those pigments (Adams and Hunter 2012). In laboratory studies, scientists have been able to induce the formation of intracytoplasmic membranes in Escherichia coli by making the cells overproduce certain membrane proteins (Jamin et al. 2018, Arechaga et al. 2000).

Ribosomes

Scattered within the cytosol of all bacteria are thousands of ribosomes. Ribosomes may also be found loosely attached to the cytoplasmic membrane (Prescott et al. 1993). These structures are responsible for the synthesis of proteins in a cell. In essence, they are molecular machines that take segments of an organism's genetic code (specified by RNA nucleotides) and "translate" them into sequences of amino acids to form polypeptides (chains of amino acids). Once a polypeptide is completed and dissociates from the ribosome, it spontaneously folds up to form a protein (with the shape of the protein determined by the sequence of amino acids in the polypeptide chain).

Ribosomes are found in all cellular domains— Bacteria, Eukarya, and Archaea. They consist of two subunits— a large subunit and a small subunit. Each of the subunits is made up of a complex of RNA and proteins. Over 50 different ribosomal proteins have been identified in bacteria; 34 of these proteins are also found in the ribosomes of archaea and eukaryotes (Yutin et al. 2012).

Ribosomes and their components are described in terms of their sedimentation values or "S values" (also known as sedimentation coefficients), which refer to their rate of sedimentation in an ultracentrifuge. S values are measured in Svedberg units (abbreviated as "S"). When particles are suspended in a liquid medium and a centrifugal force is applied to them, the rate at which they separate out to form sediment depends on their mass, volume, and shape; with heavier, more compact particles tending to move/sink to the bottom of the centrifuge tube at a faster rate (i.e., have a larger S value) than lighter, less compact particles.

Bacteria and archaea have ribosomes with a sedimentation value of 70S. The large subunit has a sedimentation value of 50S and is made up to two ribosomal RNA (rRNA) molecules (a 5S rRNA and a 23S rRNA) and approximately 34 ribosomal proteins, while the small subunit has a sedimentation value of 30S and is made up of a single rRNA molecule (a 16S rRNA) and approximately 21 ribosomal proteins (Prescott et al. 1993). In comparison, the eukaryotic ribosome is larger— 80S, and it is made up of 40S and 60S subunits (Prescott et al. 1993).

Protein synthesis is of vital importance to cells. Virtually all metabolic reactions that take place in cells involve proteins. Proteins are also involved in the transport of substances across membranes, cell signaling, the segregation of chromosomes, cell division, and the maintenance of cell shape, among other functions. The bulk of a cell's total energy budget goes towards making proteins (Harold 1986, Russel and Cook, 1995).

When bacterial cells are stressed, e.g., when they are starved of nutrients, they are able to deactivate many of their ribosomes to conserve energy—so they don't waste energy trying to make proteins under unfavorable conditions. With a substantial reduction in the number of proteins being produced, other metabolic processes within the cell (which are dependent on proteins) are likely scaled back proportionately, further conserving energy (McKay and Portnoy 2015).

The deactivation of ribosomes occurs as follows: When nutrient-limited or otherwise stressed, bacteria produce proteins known as dimerizing factors. These proteins interact with (bind to) 70S ribosomes and cause two 70S ribosomes to bind together, forming what is known as a 100S ribosome. The 100S ribosomes are nonfunctional—they are unable to produce proteins and thus are called "hibernating ribosomes." Once environmental conditions become favorable again (e.g., once starved cells are provided with fresh, nutrient-rich media) the 100S ribosomes dissociate into 70S ribosomes, which once again become active and capable of making proteins.

A bacterium's ability to produce 100S ribosomes increases its survival rate enormously— reportedly by 2 to 3 orders of magnitude (Gohara and Yap 2018). Without this ability, the ribosomes in stressed bacteria become rapidly degraded, and the cells tend to die prematurely (Basu and Yap 2017). Virtually all bacteria appear to be capable of producing 100S ribosomes, although the mechanisms involved vary depending on the type of bacterium (McKay and Portnoy 2015).

Genetic Material

The genetic material, transcription, and translation

Most bacteria have their DNA in the form of a single circular chromosome (diCenzo and Finan 2017). The DNA is double-stranded, highly condensed— i.e., linearly compacted over 1,000-fold (Holmes and Cozzarelli 2000, Trun and Marko 1998), and occurs in association with various proteins or protein complexes (Badrinarayan et al. 2015, Trojanowski et al. 2018). The associated proteins are involved in the organization, condensation, and segregation of the chromosomes and may have other functions related to the cell cycle (Badrinarayan et al. 2015, Trojanowski et al. 2018).

Bacterial DNA is densely packed with genes— it contains very few intravening sequences that don't code for proteins. Thus in bacteria, the size of the genome (the complete set of genetic material for the organism) is strongly proportional to the number of genes found in that organism (Koonin 2011). This is in contrast to eukaryotes, where the relationship between genome size and gene size is decoupled— eukaryotes tend to contain relatively large amounts of noncoding DNA, and the quantity of noncoding DNA may vary hugely depending on the species (Koonin 2011).

Unlike in eukaryotes, bacterial chromosomes are not enclosed in a special compartment within the cell. Instead, the genetic material is suspended within the cytoplasmic matrix. The area of the cell containing the chromosome(s) is referred to as the nucleoid. (The outline of the nucleoid is coincident with the outline of the chromosomal material within the cell.) The nucleoid can be thought of as an organized tangle of chromosomal material that takes the form of a mesh, which has a certain pore size (Castellana et al. 2016). Molecules smaller than the pores can freely pass into the interior of the mesh, while molecules larger than the pores cannot.

The nucleoid is typically located near the middle of the cell (Bakshi et al. 2012). In the nucleoid, RNA is produced from DNA in a process known as transcription. The types of RNA that are produced include ribosomal RNA (rRNA)— the primary component of ribosomes, messenger RNA (mRNA)— which is used as the template from which to make proteins, and transfer RNA (tRNA)— which carries amino acids to the mRNA based on the sequence of nucleotides encoded in the mRNA. Whereas rRNA and tRNA are stable forms of RNA, mRNA has a lifespan of around 6 to 7 minutes (Bernstein et al. 2002).

Unlike the case of eukaryotes, where transcription (production of RNA from DNA) and translation (synthesis of proteins) occur at separate locations and times (transcription in the nucleus and translation in ribosome-rich areas of the cytoplasm and endoplasmic reticulum), in bacteria the two processes can be coupled (in a phenomenon known as co-transcriptional translation) (Miller et al. 1970, Bakshi et al. 2015). Although co-transcriptional translation occurs within the nucleoid, the nucleoid has been found to be largely devoid of ribosomes— in part because bound ribosomes are larger than the average pore size of the nucleoid (Castellana et al. 2016). At least in the case of Escherichia coli, the ribosomes tend to be strongly localized to the cell poles and segregated from the nucleoid (Bakshi et al. 2012). However, it has been shown that free ribosomal subunits (30S and 50S subunits) can readily enter the nucleoid (Sanamrad et al. 2014). Thus the following scenario is believed to occur: as the mRNAs are formed, the ribosomal subunits bind to them, i.e., a 30S subunit binds to an initiation site on the mRNA, and then a 50S subunit binds to the 30S unit, forming a functional 70S ribosome. Multiple ribosomes bind to a single mRNA in this manner, forming what is known as a 70S polysome. Then, through some phenomenon— perhaps due at least in part to excluded volume effects— the 70S polysomes are excluded from the nucleoid and migrate towards the poles of the cell (Castellana et al. 2016, Bakshi et al. 2015). Protein synthesis continues outside of the nucleoid; the mRNAs can be translated into proteins repeatedly before finally degrading (Bakshi et al. 2015).

At least with regards to Escherichia coli, there is a cycling of ribosomes within the cell: ribosomal subunits flow into the nucleoid where they bind to mRNA to form 70S polysomes; the polysomes flow out of the nucleoid towards the cell poles where proteins synthesis is continued; then at the poles the mRNAs degrade and the ribosomes dissociate into subunits, which flow into the nucleoid (Bashi et al. 2015, Castellano et al. 2016). Based on studies with Escherichia coli, most protein synthesis occurs post-transcriptionally, outside of the nucleoid (Bashi et al. 2015, Castellano et al. 2016), while around 10 to 15 percent of protein synthesis appears to occur co-transcriptionally, within the nucleoid (Bashi et al. 2015).

DNA replication

The bacterial chromosome consists of a single replicon—it has a single origin of replication (replication is initiated from just one point on the chromosome) and the whole chromosome is replicated as a single unit. From the origin of replication (known as oriC), replication of the DNA proceeds in two directions around the chromosome. Starting at the origin, the two strands of the original DNA begin to separate, forming a "bubble" and new DNA is synthesized along each of the original strands. As replication proceeds, the "bubble" expands in both directions until the entire chromosome is copied (Figure 3).

In contrast to bacteria, eukaryotes have linear chromosomes with multiple origins of replication— they can potentially have hundreds of replication origins per chromosome (Kuzminov 2014). On eukaryotic chromosomes, origins of replication occur every 10 to 100 μm and each of the associated replicons can be copied simultaneously, so that a large DNA molecule may be quickly replicated (Prescott et al. 1993). Because bacterial chromosomes have only a single origin of replication, replication time increases with the size of the chromosome. Thus, it is advantageous for bacteria to have small chromosomes so that they can maximize their rate of reproduction. ^[1]

Although most bacteria have circular chromosomes, linear chromosomes are found in some taxa, such as Borreliella (Borrelia) species, which cause lyme disease (Ferdows and Barbour 1989, Casjens et al. 1995), and several species of Streptomyces (Yang et al. 2002), which are the source of many of our antibiotics (Chater 2006). Linear chromosomes in bacteria are apparently evolved from circular bacterial chromosomes (Volff and Altenbuchner 2000). Thus, in contrast to eukaryotic chromosomes, linear chromosomes in bacteria have only one origin of replication (Trojanowski et al 2018). The origin is typically located near the middle of the chromosome (Volff and Altenbuchner 2000, Picardeau et al. 1999, Hopwood 2006).

The cell cycle

The cell cycle in bacteria and other organisms involves, among other things, the replication of the chromosome(s), the segregation of the daughter chromosomes to different ends of the cell, and cell division— the splitting apart of the cell into daughter cells (with each daughter cell receiving a copy of the genetic material). Whereas eukaryotes have cell cycles with discrete, non-overlapping stages, bacterial cell cycles are quite different.

Compared to eukaryotes, bacteria are unusual in that they have cell cycles in which DNA replication and chromosome segregation occur simultaneously (Kuzminov 2013, Badrinarayanan et al. 2015). Shortly after a given locus on a chromosome is replicated, the sister loci are segregated; so the segregation of the various loci along the chromosome occurs sequentially rather than simultaneously, and the overall rate of segregation equals the rate of replication (Kuzminov 2013). In addition, in at least some taxa of bacteria multiple rounds of chromosome replication can occur in parallel within a single cell cycle in a phenomenon known as multifork replication (Yoshikawa et al. 1964, Kuzminov 2013, Bremer and Dennis 1996, Trojanowski et al. 2017). In other words, shortly after a cell has begun replication to create daughter chromosomes (and before the daughter chromosomes are fully copied and fully segregated into daughter nucleoids) replication of the daughter chromosomes may commence (to produce granddaughter chromosomes). And shortly after replication of the granddaughter chromosomes has started (and before the daughter and granddaughter chromosomes have been fully replicated and segregated), the cell can begin replication of the granddaughter chromosomes (to produce great-granddaughter chromosomes), etc. In Escherichia coli, as many as four overlapping rounds of chromosome replication can occur (Morigen et al. 2009). Multifork replication tends to occur in bacteria that are growing rapidly under ideal, nutrient-rich conditions (Wang and Levin 2009). In slowly growing cells of Escherichia coli, the chromosome is only replicated once prior to cell division (Cooper and Helmstetter 1968, Skarstad et al. 1983). However, multifork replication has been observed in a fraction of slow-growing Mycobacterium smegmatus cells, including cells grown under suboptimal conditions (Trojanowski et al. 2017). So the interplay of factors causing multifork replication is not fully known. Whether or not multifork replication occurs may depend on the growth rate (Wang and Levin 2009), the taxa of bacteria (Fossum et al. 2007), and other factors.

The phenomenon of multifork replication has some interesting consequences: (1) The number of copies (copy number) of the chromosome (ploidy) can change from one generation to the next— depending on how fast the cells are growing and what stage of growth they are in (Pecoraro et al. 2011, Maldonado et al. 1994); (2) Cells can inherit incomplete copies of chromosomes (i.e., copy numbers that are not whole numbers). For example, when growing under optimal conditions, Escherichia coli is merodiploid (having one full copy plus one or more partial copies of its chromosome) to mero-oligoploid (having a few full copies and one or more partial copies of its chromosome) (Pecoraro et al. 2010); and (3) In stark contrast to eukaryotic cells —where the time required to complete the cell cycle is equal to the sum of the amounts of time required to complete each of the discrete cell cycle stages— many bacteria that undergo multifork replication can divide faster than the amount of time it takes to replicate their chromosome. For example, Escherichia coli can divide as quickly as once every 24 minutes whereas it takes 42 minutes to over an hour to replicate its chromosome (Bremer and Dennis 1996).

The bacterial cell cycle varies somewhat depending on the taxa of bacteria. Bacteria with nontypical cell cycles include species with more than one chromosome/essential replicon, those that undergo cell differentiation, and species with complicated life cycles (Trojanowski et al. 2018). The cell cycle in bacteria is still being elucidated. It is described by some as appearing to consist of "a set of coordinated but independent events"—as opposed to one unified process (Wang and Levin 2009)— and by others as a single event, with the different cell cycle processes occurring together, in the same location, probably by physically interacting entities (Kuzminov 2013).

Chromosomes and other replicons within the cell

Chromosome (Primary Chromosome, Primary Essential Replicon).

The bacterial chromosome is sometimes referred to as the primary chromosome or the primary essential replicon, because other types of replicons—secondary replicons— may be present in a cell. (All DNA molecules found within bacterial cells [i.e., chromosomes and secondary replicons] consist of single replicons [i.e., having one origin of replication]— this is in contrast to eukaryotic chromosomes, which are made up of many replicons.)

The chromosome is most easily distinguished from secondary replicons based on size: the chromosome is always the largest replicon in the cell (Harrison et al. 2010, diCenzo and Finan 2017). It contains all or most of the essential genes needed by the bacterium for survival (Harroson et al. 2010, diCenzo and Finan 2017, Ochman 2002, Egan et al. 2005), and its replication is tied to the cell cycle (Egan et al. 2005).

Secondary replicons

Secondary replicons include plasmids, megaplasmids, and chromids (diCenzo and Finan 2017). One or more of these types of secondary replicons may be found within a cell. Based on available genome data, approximately 40 percent of bacterial species contain secondary replicons (diCenzo and Finan 2017). Like chromosomes, secondary replicons consist of double-stranded DNA molecules, which are typically in the form of a closed circle, although some are linear (Ochman 2002, Hopwood 2006, Chaconas and Kobryn 2010). Secondary replicons have a replication system that is distinct from that of the chromosome; they have a plasmid-like replication system with a characteristic plasmid-like origin of replication (Fournes et al. 2018). Secondary replicons that are greater than 25 kilobases in size tend to carry genes for partition systems (Planchenault et al. 2020). Partition systems segregate copies of secondary replicons, so that when a cell divides, each daughter cell will receive a copy (or copies) of the replicon. Partition systems are generally absent from secondary replicons less than 25 kilobases (Planchenault et al. 2020); with no active partition system, these smaller replicons are randomly segregated into daughter cells. (When random segregation occurs, there is no guarantee that both daughter cells will receive a copy of the replicon.) Secondary replicons are described below:

• plasmids — are small DNA molecules that are considered to be dispensible to the cell; they don't carry any core bacterial genes essential for the survival/viability of their host (Egan et al. 2005). A plasmid is an example of an acellular parasite. It is an independent agent that utilizes the host cell's machinery to replicate its DNA and, in many cases, to spread itself to other host cells. Plasmids are replicated independently of the host cell's chromosome and independently of the cell cycle (del Solar et al. 1998 referenced in Egan et al. 2005).^[2] A plasmid's DNA typically includes core plasmid genes— for replication of the plasmid and transmission of the plasmid to other bacteria, and accessory genes— other miscellaneous genes, which in some cases may prove beneficial to the bacterial host, for example by providing antibiotic resistance or resistance to heavy metals (Maclean and San Millan 2015). Plasmids that lack partition systems (i.e., plasmids < 25 kilobases) are normally found along the outer edge (just outside) of the nucleoid (they appear to be excluded from the nucleoid), while plasmids that have partition systems are found within the volume of the nucleoid (but separate from the bacterial chromosome) (Planchenault et al. 2020).^[3]

• megaplasmids— like plasmids, megaplasmids don't carry any genes needed by the bacterium for survival/viability. As is true for larger sized plasmids, megaplasmids are found within the volume of the nucleoid and they contain active partition systems. Megaplasmids are distinguished from plasmids solely based on size; however, there is no standardly accepted size cutoff between megaplasmids and plasmids.^[4] Although they are essentially defined as large plasmids, megaplasmids appear to have genomic features and other characteristics that are intermediate between those of plasmids and chromids (diCenzo and Finan 2017). In contrast to plasmids, megaplasmids may replicate and segregate in coordination with the bacterial cell cycle (diCenzo and Finan 2017).

• chromids (Secondary Chromosomes, Secondary Essential Replicons)—are so named because they contain characteristics of both plasmids and chromosomes. Unlike plasmids and megaplasmids, chromids contain one or more core bacterial genes essential for the survival/viability of the bacterium; however, chromids contain fewer of these essential genes than the chromosome (Egan et al. 2005). Chromids are generally larger (consist of more base pairs) than megaplasmids, although their sizes tend to overlap (diCenzo and Finan 2017). Like megaplasmids, chromids appear to replicate and segregate in coordination with the host's cell cycle (diCenzo and Finan 2017) and they have active partition systems (Planchenault et al. 2020). And, apparently because they possess a partition system, they are able to reside within the volume of the nucleoid (Planchenault et al. 2020), where they may take advantage of entropic flow to segregate copies of themselves into daughter cells (Planchenault et al. 2020). Often chromids have genes for specialized functions that may be beneficial to bacteria under certain environmental conditions (Egan et al. 2005).

Evolution of secondary replicons

It is commonly believed that these three types of secondary replicons represent an evolutionary continuum— from recently acquired DNA (in the form of a plasmid), to DNA that's stably or nearly stably incorporated into the host's genome (in the form of a chromid).

Plasmids are replicons that were apparently obtained fairly recently, from another bacterium or other organism by means of horizontal gene transfer. (Horizontal gene transfer includes a number of processes by which genes are transmitted from one individual to another by some means other than by inheritance from a parent.) Through horizontal gene transfer, bacteria have the potential to rapidly adapt to new or changing environmental conditions—they can instantly obtain genes needed to survive in a new environment from some other organism that already has those genes, rather than wait for the needed genes to evolve over time and over many, many generations through mutations.

A plasmid that contains no genes that benefit a bacterium, will impose a fitness cost on its bacterial host (MacLean and San Millan 2015). A bacterium carrying such a plasmid needs to expend energy and resources to replicate, transcribe and translate the plasmid's genes. Organisms generally don't have unlimited energy and resources. Therefore, a bacterium carrying a parasitic plasmid, will have less energy and resources available to devote to its own growth and reproduction. Consequently, an infected bacterium will not be able to effectively compete with similar (plasmid-free) bacteria, and ultimately the plasmid-carrying bacterium will produce fewer offspring. Thus there is a strong evolutionary tendency for nonbeneficial plasmids to be lost from bacteria over time (unless the plasmids can spread themselves to other bacteria at a rate faster than the bacteria can lose the plasmids).

If on the other hand, a plasmid contains one or more genes that benefit the bacterium— the plasmid functions as a symbiont rather than a parasite—there will be a tendency for the plasmid (or at least the plasmid's beneficial genes) to be retained over time. The beneficial genes may enable the bacterium to outcompete other bacteria or to colonize a novel environment or niche where few or no competitors exist.

When a foreign DNA molecule such as a plasmid is taken into a cell, initially it has a very different nucleotide composition than that of the host cell's chromosome. However, over long periods of time, the foreign DNA becomes increasingly "domesticated" ("ameliorated") so that its nucleotide composition (genetic signature) begins to closely resemble that of the host cell's chromosome (Lawrence and Ochman 1997). For example, the percentage of the bases guanine and cytosine (G+C) making up the DNA, and the dinucleotide relative abundance become more similar to that of the host chromosome (diCenzo and Finan 2017). These changes in the foreign DNA are believed to occur because the host cell has a very different internal environment compared to the environment in which the foreign DNA formerly occurred. The unique environment of the host cell exerts certain directional mutation pressures—the DNA in the cell is affected by mutational biases— so that over time the DNA takes on a characteristic genomic signature (Sueoka 1988, Lawrence and Ochman 1997, Suzuki et al. 2008). Differences in genomic signatures (e.g. percentage of G+C) can be used to estimate how long foreign DNA has resided in a cell (Lawrence and Ochman 1997) or to determine whether bacteria are closely related to one another (Sueoka 1962).

Most scientists believe that chromids evolved from plasmids (i.e., from megaplasmids). Some of the lines of evidence in support of this theory include the plasmid-like replication machinery of secondary replicons (including chromids) [Fournes et al. 2018], as well as evidence from genomic signature data. Chromids have genomic signatures (GC content and dinucleotide relative abundances) that are very similar to those of their respective host chromosomes (e.g., GC content differing by less than 1% [diCenzo and Finan 2017]). Compared to chromids, the genomic signatures of megaplasmids are less similar to those of the chromosome, and the genomic signatures of plasmids are even more different from the chromosome (diCenzo and Finan 2017). Thus the data suggest that chromids have resided in cells for a relatively long evolutionary time period, that megaplasmids have been present in cells for a somewhat shorter period of time, and that plasmids were acquired fairly recently.

For a plasmid to evolve into a chromid, not only does "genomic amelioration" need to occur, but the plasmid must also acquire one or more essential (core) bacterial genes. Although the phenomenon of genomic domestication/amelioration is well known (Suzuki et al. 2008), scientists don't have a clear understanding of how plasmids/chromids acquire essential bacterial genes.

Plasmids and chromosomes may carry other acellular parasites— for example genetic elements known as transposons, which have the ability to "jump" from one DNA molecule to another. (The "jumping" involves excision of the DNA sequence from the original site [or duplication of the DNA sequence] followed by insertion into the new location). The beneficial genes carried by plasmids are often found in transposons or other mobile genetic elements (MacLean and San Millan 2015). Thus, it is possible for genes to move— via mobile genetic elements— from plasmid to chromosome or vice-versa.

It is also known that one or more secondary replicons may fuse with the host chromosome, creating a single replicon from the cointegrated DNA molecules (Guo et al. 2003, Val et al. 2014).^[5] The fused DNA molecule (called a cointegrate) can split back into its original replicons, as long as the splits occur at the same sites where the molecules were fused; this appears to be what typically occurs when cointegrates break apart (Guo et al. 2003). However, if the splits occur at other sites— sites within the original replicons rather than between them— new replicons may be created with novel combinations of genes (Guo et al. 2003). Thus if a chromosome and a plasmid fused together and then split apart, it may be possible that one or more core genes from the chromosome could end up on the plasmid (and one or more plasmid genes could end up on the chromosome (Poirion and Lafay 2018).

Another way that an essential core bacterial gene, formerly located on a chromosome, could end up on a plasmid is if the cell acquired a new copy of the gene on the plasmid/chromid (in addition to the existing copy on the chromosome) (diCenzo and Finan 2017). Then if the copy of the gene was lost from the chromosome (e.g., due to a detrimental mutation), the cell could still survive, relying on the copy on the chromid. Gene redundancy could potentially occur if a core gene on the chromosome was duplicated and a copy ended up on a plasmid, or if the cell acquired— through horizontal gene transfer— a plasmid with a copy of the core gene (or a functionally equivalent gene) (diCenzo and Finan 2017).

Multipartite genomes

Bacteria containing two or more large DNA molecules—i.e., a chromosome and one or more megaplasmids and/or chromids—are said to have a "multipartite" genome. Based on available genome data, around 10 percent of bacterial species have multipartite genomes (Harrison et al. 2010, diCenzo and Finan 2017). Bacteria with multipartite genomes include species that interact with eukaryotes— as pathogens or symbionts, species found in polluted environments that are capable of catabolizing pollutants, and species that occur in extreme environments (diCenzo and Finan 2017). However, not all bacteria occupying these niches have multipartite genomes (diCenzo and Finan 2017). Nonetheless, those that do apparently acquired genes from plasmids enabling these bacteria to thrive in new environments. Bacteria found in the same environments that don't have multipartite genomes may have also acquired their niche-specific genes via horizontal gene transfer, but from a different acellular parasite.

Scientists have recently begun to recognize the prevalence of genetic elements known as integrative and conjugative elements (ICEs), which, like plasmids, appear to be important in enabling bacteria to quickly adapt to new niches. Like plasmids, ICEs are capable of spreading copies of themselves from one host cell to another. ICEs spread to new host cells in the same manner as plasmids— via a process known as conjugation. Also like plasmids, ICEs often carry genes that may enhance the fitness of their bacterial hosts. ICEs differ from plasmids in that they normally integrate themselves into the host cell's chromosome (rather than remaining separate from it). However, the distinction between these two types of genetic elements is not always sharp and over time one type may give rise to another (Guglielmini et al. 2011). Scientists have tended to overlook ICEs because they are difficult to identify and delimit from sequenced bacterial genomes (Ambroset et al. 2015, Cury et al. 2017). However, given their apparent importance in microbiology, ICEs are increasingly being studied.

Cell Envelope

The bacterial cell envelope consists of cell membrane, cell wall, and other materials that surround the cytoplasm. The actual layers making up the cell envelope vary depending on the species of bacterium. All cells, at a minimum, have a cytoplasmic membrane. There are two common cell envelope types— gram positive and gram negative. These envelope types have traditionally been determined using a staining technique known as gram staining, which is still used today.

The gram staining procedure was developed over 130 years ago by the Danish doctor Hans Christian Gram (Gram 1884). Dr. Gram noticed that different types of bacteria vary in their ability to retain dyes. He tested whether bacteria retained the dye crystal violet by

1. Applying the crystal violet dye to a heat-fixed smear of bacteria on a glass slide;
2. Washing off the excess dye using water;
3. Applying iodine in order to set or "fix" the dye;
4. Rinsing with alcohol to wash off the dye; and
5. Rinsing with water to stop the action of the alcohol.

Dr. Gram found that certain bacteria retained the violet-colored dye after this procedure (the gram-positive bacteria), while other bacteria did not retain the dye (the gram-negative bacteria). ^[6]

Although Dr. Gram recognized that bacteria could be categorized into two types based on their ability to retain the crystal violet stain, he was unaware of the significance of his finding and did not have knowledge of the physical differences between the two types of bacteria (Sandle 2004).

Today we know that gram-positive- and gram-negative-staining bacteria have very different cell envelopes.

Gram-positive Cell Envelope

A classical gram-positive cell envelope consists of the following layers (listed from interior to exterior):

1. A cytoplasmic membrane (also referred to as the cell membrane or plasma membrane), consisting of a symmetric bilayer of phospholipids with embedded proteins. This membrane is around 5 to 10 nanometers thick (Prescott et al. 1993). It encloses the cytoplasm and controls the movement of substances into and out of the cytoplasmic compartment. It is also the site of various biochemical processes. The electron transport chains involved in respiration and photosynthesis are located here.

   Bacterial cells maintain an electrochemical gradient across the cytoplasmic membrane. (There is a lower concentration of protons inside the cytoplasmic compartment than outside of it.) A multi-subunit protein complex called ATP synthase is embedded in the cytoplasmic membrane. ATP synthase uses the potential energy of the transmembrane gradient to make ATP (a ready-to-use source of energy for the cell).

   

2. Outside of the cytoplasmic membrane is a relatively thick cell wall, containing multiple layers of peptidoglycan, with a total thickness ranging from 30 to 100 nm (Silhavy et al. 2010). In gram positive bacteria the peptidoglycan layer is usually complexed with polymers of teichoic acids, which may constitute a substantial portion of the cell wall's mass (Brown et al. 2013, Silhavy et al. 2010). Two types of teichoic acids are present— wall teichoic acids, which are attached to the peptidoglycan and extend beyond the peptidoglycan layer (to the outermost reaches of the cell wall), and lipoteichoic acids, which are attached to the cytoplasmic membrane and extend into the peptidoglycan layer (Brown et al. 2013). Teichoic acids are negatively charged and cause the gram-positive cell wall to have an overall net negative charge (Prescott et al. 1993, Jordan et al. 2007). These polymers have many functions in the cell, which may vary depending on the species of bacteria (Silhavy et al. 2010). Wall teichoic acids appear to play a role in cell morphology and division, autolytic activity, ion homeostasis, tolerance of high temperatures and osmotic stress, cell adhesion and biofilm formation, and defense against antibiotics (Brown et al. 2013).

   Recently, the gram-positive cell wall has been found to comprise two zones: a high density outer wall zone and a low density inner wall zone (Matias et al. 2005). The outer wall zone corresponds to the peptidoglycan layer of the cell wall, while the inner wall zone consists of a space located between the cytoplasmic membrane and the outer wall zone (Zuber et al. 2006, Matias and Beveridge 2005). The inner wall zone is sometimes referred to as the periplasmic space, although that term is more commonly used to refer to the space/compartment between two membranes (Forster and Marquis 2012).

Gram-negative Cell Envelope

In contrast to the classical gram positive cell, the classical gram negative cell has (1) a relatively thin cell wall, (2) an additional membrane—called the outer membrane— located outside of the cell wall, and (3) a fluid-filled compartment called the periplasmic space or periplasm, which is bounded by the two membranes of the cell.

The layers found in a classical gram-negative cell envelope are listed and described below:

1. A cytoplasmic membrane (also referred to as the inner membrane, cell membrane, or plasma membrane). It has the same structure, composition, and functions as the cytoplasmic membrane found in gram-positive bacteria.

2. A relatively thin cell wall, containing one to a few layers of peptidoglycan, with a thickness of 2 to 6 nm (Silhavy et al. 2010, Hoiczyk and Hansel 2000). Unlike the cell walls of gram-positive bacteria, gram-negative cell walls lack teichoic acids (Cheng and Costerton 1977, Prescott et al. 1993). The cell wall is attached to both the cytoplasmic membrane and the outer membrane by membrane-anchored proteins (Kovacs-Simon et al. 2011), with some protein complexes extending across both membranes (Nikaido 1996).

3. An outer membrane, around 7.5 to 10 nm thick (Salton and Kim 1996), consisting of an asymmetric lipid bilayer. This membrane screens out chemicals that are harmful to the cell and adds structural stability. The inner leaflet of the membrane is made up of phospholipid molecules, while the outer leaflet normally consists of glycolipids— typically lipopolysaccharide (LPS) molecules (Galdiero et al. 2012). Approximately 50 percent of the outer membrane is made up of proteins (Koebnik et al. 2000). Two main types of proteins are found in outer membranes: lipoproteins and transmembrane beta-barrel proteins.

   

   Lipoproteins are found in both gram-negative and gram-positive bacteria and occur in both cytoplasmic and outer membranes (Kovacs-Simon et al. 2011). They may be found in the outer leaflet of the cytoplasmic membrane, the inner leaflet of the outer membrane, and/or the outer leaflet of the outer membrane (Kovacs-Simon et al. 2010). They do not span both layers of the membrane. These proteins have a lipid component, which apparently aligns with and anchors them (via hydrophobic interactions) to the fatty acid tails of the phosopholipid and LPS molecules in the membrane (Kovacs-Simon et al. 2011). Lipoproteins in the outer membrane are involved in cell wall synthesis, secretion systems, and antibiotic efflux pumps (Grabowicz and Silhavy 2017). One type of lipoprotein, called Braun's lipoprotein (murein lipoprotein or Lpp), attaches the peptidoglycan wall to the outer membrane. Additional proteins are apparently involved in a network that links together the cell wall and the outer membrane; together the Braun's lipoprotein and the other proteins in the network appear to be of critical importance in maintaining the integrity of the gram-negative cell envelope (Dramsi et al. 2008).

   Beta-barrel transmembrane proteins are characteristic components of the gram-negative outer membrane. These proteins (sometimes referred to as outer membrane proteins) are not found in the cytoplasmic membrane or in any other cellular membrane (except for the outer membranes of mitochondria and chloroplasts— two eukaryotic organelles that apparently evolved from gram-negative bacteria) (Koebnik et al. 2000, Fairman et al. 2011).

   A common type of beta-barrel protein found in outer membranes are porins. They are so-named because they form pores or channels in the membrane. The pores are filled with water and allow small hydrophilic molecules to diffuse across the membrane (Galdiero et al. 2012). General/nonspecific porins are abundant in outer membranes and allow many different types of ions and polar molecules to pass through (as long as the diffusing entities are small enough to fit through the channel), with some selectivity for either cations or anions depending on the porin (Galdiero et al. 2012). Less common are substrate-specific porins (i.e., porins that only allow a specific molecule to pass through— for example sucrose-specific porins). Substrate-specific porins are expressed at increased levels when needed (their synthesis is induced). These porins have channel interiors with features that make it easier for particular molecules to diffuse into the cell (Forst et al. 1998). Besides their role in nutrient exchange across the outer membrane, porins may also be involved in pathogenicity (Galdiero et al. 2012).

   In addition to porins, other types of beta-barrel proteins found in outer membranes include proteins involved in the active transport of large substrates into the cell (some of these proteins are also known to play a part in signal transduction^[7]), proteins involved in the active transport of molecules out of the cell; those involved in the secretion of proteins (primarily virulence factors); ones that function as enzymes; and proteins with structural functions (Kim et al. 2012, Koebnik et al. 2010).

   Unlike the cytoplasmic membrane, the outer membrane is not an energized membrane. No gram-negative bacterium is known to have an electrochemical gradient across its outer membrane and ATP synthase has never been found in bacterial outer membranes (Küper et al. 2010). However, active transport does occur across the outer membrane; the energy required for this transport is ultimately obtained from the electrochemical potential of the cytoplasmic membrane (Braun et al. 1996).

4. A periplasmic space or periplasm—a compartment located between the cytoplasmic membrane and the outer membrane. (The cell wall extends through this space.) Like the cytoplasm, the periplasm is a viscous fluid-filled compartment. It may constitute 7 to 40 percent of the total cell volume (Kulp and Koehn 2010). Found within this compartment are binding and trafficking proteins, various enzymes (including degradative and detoxifying enzymes), secreted materials, and components for synthesis of the cell wall and outer membrane (Beveridge 1999). The periplasm is devoid of ATP and other molecular energy sources (Kulp and Kuehn 2010).

Although both gram-negative and gram-positive species can be highly pathogenic (depending on the species), gram-negative bacteria have some special characteristics associated with their outer membrane that may enhance their pathogenicity, as summarized below:

• The outer membrane provides gram-negative bacteria with an extra layer of protection— an additional selective barrier— against antibiotics, toxins, and other harmful substances. In general, gram-negative species are more resistant to antibiotics than gram-positive species, at least in part because of their extra membrane (Silhavy et al. 2010, Nikaido 1996). The outer membrane is impermeable to large charged molecules (Galdiero et al. 2012), and due to the presence of LPS, provides an effective barrier against hydrophobic molecules (Silvahy et al. 2010). Hydrophobic molecules diffuse across the outer membrane about two orders of magnitude slower than across a typical cytoplasmic membrane (Nikaido 2003). This is a benefit because most clinically important antibiotics have some degree of hydrophobicity (Cohen 2004). Although both gram-positive and gram-negative bacteria have efflux pumps that pump antibiotics and other toxins out of the cell, these pumps tend to be more effective in gram-negative bacteria because their cell envelopes are less permeable (Fernández and Hancock 2012)— they can pump out the toxins faster than the toxins can enter the cell.

  

• Outer membranes contain LPS molecules, which are known endotoxins. The toxin is released when the bacterial cells die and break open. Small amounts of LPS are also released from live cells, as a by-product of the normal, on-going maintenance and renewal of the outer membrane (Wassenaar and Zimmermann 2018) and during cell division (Matsuura 2013). Virtually all gram-negative species contain LPS in their outer membranes.^[8] In mammals, LPS stimulates the immune system and can induce fever and inflammation (Wassenaar and Zimmermann 2018). If a gram-negative bacterial infection spreads to the bloodstream, septic shock may occur (due to an overzealous inflammatory response), potentially resulting in organ failure and death (Wassenaar and Zimmermann 2018).^[9]

  LPS molecules contain three main components: (1) Lipid A— a phosphorylated diglucosamine molecule with fatty acid tails, which anchors the LPS into the membrane; (2) a "core" oligosaccharide, which is attached to Lipid A; and (3) a relatively long O-antigen polysaccharide, which is connected to the core oligosaccharide and extends out from the cell exterior into the surrounding medium (Raetz and Whitfield 2002, Nikaido 2003, Clifton et al. 2013). The toxic part of LPS is the Lipid A component (Raetz and Whitfield 2002, Wassenaar and Zimmermann 2018, Stewart et al. 2016).^[10] Lipid A is the portion of the molecule that is recognized by the innate immune system; thus the presence of lipid A alone in the bloodstream can lead to septic shock (Ramachandran 2014, Raetz and Whitfield 2002). However, the adaptive immune system— which launches its attacks at cells containing specific antigens— usually directs its response based on the composition of the O-antigen (Bryant et al. 2010).

• The o-antigen is the most variable part of the LPS molecule (Lerouge and Vanderleyden 2001, Matsuura 2013, Stewart et al. 2006). Variability in the o-antigen may enable some bacteria to evade the host's immune response (Raetz and Whitfield 2002, Nikaido 2003), resulting in persistent infections. The immune systems of mammals and other vertebrates are able to distinguish their own cells from those of foreign invaders. They recognize the presence of a foreign invader based on a macromolecule (e.g. a protein or a polysaccharide) that occurs in association with that invader, typically on the surface of the invader's cell. Such a macromolecule—which is recognized by the immune system and elicits an immune response—is called an antigen. The first time a host is exposed to a foreign antigen it can take several days for the immune system to launch a response that specifically targets the pathogen containing that antigen (Alberts et al. 1994). (A nonspecific immune response happens right away but is generally less effective.) However, the immune system retains a memory of the antigen after the infection has passed, and upon subsequent infection with the same pathogen/antigen the immune system is able to quickly launch a strong targeted attack. When an antigen, such as the o-antigen, is highly variable and changes its structure and/or composition frequently it becomes much harder for the immune system to clear the infection. Once the antigen changes its form, the T-cells, B-cells, antibodies, and memory cells that were once specific to that pathogen are no longer effective in fighting the infection. Each time the antigen changes, a new adaptive immune response needs to be launched from scratch.

  In some cases a single population of bacteria may contain cells that express different o-antigens (Lerouge and Vanderleyden 2001). Thus to control a single infection, a host may need to produce immune cells and antibodies specific to each type of o-antigen present.

  Certain pathogenic bacteria may evade the host's immune response by producing o-antigens (or other types of antigens) that closely resemble or "mimic" macromolecules found on the surface of the host's own cells (Matsuura 2013, Lerouge and Vanderleyden 2001). For example, Helicobacter pylori, a species commonly found in the human stomach, may express o-antigens that mimic blood group structures found on the surface of cells in the gastric mucosa (Appelmelk et al. 1996). This phenomenon, known as "molecular mimicry," may play a role in autoimmune disorders (Cusick et al. 2012).

  

  

  

• All or virtually all gram-negative bacteria are known to produce membrane vesicles from their outer membrane (Beveridge 1999, Kulp and Kuehn 2010)— they are said to "bleb" membrane vesicles off their outer membrane. These vesicles, which are released to the outside environment, may contribute to the pathogenicity of their associated bacteria.^[11] Outer membrane vesicles are generally spherical and have a diameter of around 10 to 500 nm (Domingues and Nielsen 2017). They start off as small pockets that form in the outer membrane. The pockets then pinch off to form vesicles (Figure 10). In some cases huge numbers of vesicles may bleb off an outer membrane at once (Beveridge 1999). An outer membrane vesicle contains periplasm (and whatever constituents are present in the periplasm) and is bounded by a lipid bilayer membrane. The membrane surrounding a membrane vesicle closely resembles the bacterium's outer membrane— it contains an inner leaflet of phospholipids, an outer leaflet of LPS molecules, as well as outer membrane proteins (Beveridge 1999). Outer membrane vesicles are enriched in certain proteins and lipids relative to the outer membrane from which they were formed; thus it is likely that they are produced by some specific cellular mechanism rather than from random blebbing of the membrane (Kulp and Kuehn 2010). Researchers have found that greater numbers of outer membrane vesicles are produced in response to increased stresses on the cell envelope (McBroom and Kuehn 2007).

  Outer membrane vesicles may serve as vessels to carry substances to other cells. These substances may include enzymes that break down cell walls, toxins (including the endotoxin LPS), enzymes that break down antibiotics, or even plasmids that contain genes for antibiotic resistance (Beveridge 1999, Kadurugamuwa and Beveridge 1996, Ciofu et al. 2000, Dorward et al. 1989). Consequently, membrane vesicles are capable of attacking (dissolving the cell walls of) bacterial competitors (presumably to release nutrients under poor growth conditions), they may transport and release toxins that weaken host defenses, deliver antibiotic-degrading enzymes to bacterial cells that may lack such enzymes (or deliver these enzymes to host tissues to protect infecting bacteria), and they may spread genes for antibiotic resistance (Beveridge 1999).

  The means by which outer membrane vesicles deliver substances to other cells depends on the type of recipient cell. Outer membrane vesicles attack gram-positive cells by binding to the cell wall. The membrane vesicles then break open and release enzymes that degrade the recipient's cell wall (Kadurugamuwa and Beveridge 1996). When they contact the outer membrane of gram-negative bacteria, membrane vesicles fuse to the membrane, releasing the contents of the vesicles into the recipient cell's periplasm (Beveridge 1999, Kadurugamuwa and Beveridge 1996). Outer membrane vesicles deliver their contents to eukaryotic cells either by fusing to the cell's membrane (and releasing the contents into the cell's cytoplasm) (Jäger et al. 2015) or they may be taken into a cell by endocytosis (Vanaja et al. 2016, Sharp et al. 2011, Furuta et al. 2009).

  In addition to transporting and delivering substances, outer membrane vesicles may absorb or adsorb toxins or other harmful agents, protecting bacterial cells from chemical or viral attack (Manning and Kuehn 2011, Ciofu et al. 2000). They may also play a role in biofilm formation and thus help bacteria colonize surfaces and survive in harsh environments (Schooling and Beveridge 2006, Yonezawa et al. 2009). Additionally, membrane vesicles may be used to package and remove harmful substances from the cell envelope, including misfolded proteins (McBroom and Kuehn 2007).

Monoderm vs. Diderm Bacteria

Bacteria that have one membrane (a cytoplasmic membrane only) are said to be monoderm (having "one skin"), while bacteria with two membranes (a cytoplasmic and an outer membrane) are described as being diderm (having "two skins"). The gram-staining technique is a quick and easy way to determine whether a bacterium is monoderm (which typically stain gram positive) or diderm (which typically stain gram negative), but the technique is not foolproof. Not all bacteria have a classicial gram-positive or a classical gram-negative cell envelope. Some monoderm bacteria have atypically thin cell walls and stain gram negative, and some diderm bacteria have unusually thick cell walls and stain gram positive. Culture conditions may also affect the gram stain result. For example, monoderm bacteria obtained from cultures that are old or are otherwise stressed have a tendency to stain gram negative (Beveridge 1990, Benson 1990). Some bacteria— even under ideal conditions— stain in a variable manner. They may stain gram positive or gram negative depending on their phase of growth (Beveridge 1990). Thus, in order to determine with certainty whether a bacterium is monoderm or diderm, an alternative method should be used (such as one involving transmission electron microscopy).

Which came first — monoderm or diderm bacteria?

An interesting question in biology is whether the ancestral bacterium was monoderm or diderm. In short, we don't know. Reconstructing phylogenies for ancient events is notoriously difficult (Gribaldo et al. 2010) due to increased noise, decreased evolutionary signal, and consequently a greater number of artifacts confounding the analysis (Gouy et al. 2015). Scientists cannot determine with certainty how the tree of life is rooted— for example, whether Bacteria evolved first and gave rise to Archaea or whether an ancestral cell of some other type (which no longer exists) gave rise to both Bacteria and Archaea. Scientists also can't say with confidence how Bacteria are rooted or identify the lineages that form its earliest branches (Antunes et al. 2016).

Most bacteria alive today are diderms. The known exceptions include bacteria from three phyla: Actinobacteriota, Chloroflexota, and Firmicutes. The Actinobacteriota and the Firmicutes are unusual among the bacterial phyla in that they contain both monoderm and diderm species. Most of the taxa making up these phyla are monoderm. However, one suborder of the Actinobacteriota (the Corynebacterineae) consists of bacteria with an outer membrane of mycolic acid, and at least two classes of Firmicutes contain bacteria with outer membranes of LPS— the Negativicutes and the Halanaerobiia (Halanaerobiales).

Results of a study conducted by Antunes et al. (2016) strongly suggest that, at least in the case of the Firmicutes, the ancestral bacterium was diderm (with an outer membrane containing LPS), and in multiple instances, monoderm bacteria evolved from diderm ancestors by losing the outer membrane. The authors found that both the Negativicutes and the Halanerobiia are more closely related to monoderm relatives than they are related to each other. Yet representatives from both of these diderm classes contain a large cluster of genes associated with the outer membrane that is highly conserved among the two groups— thus it appears that this large gene cluster was inherited from a common (diderm) ancestor.^[12]

It is very unlikely that the Negativicutes and/or the Halanerobiia obtained the outer membrane gene cluster via horizontal gene transfer. The outer membrane is a complex structure, and given the size of the gene cluster in these taxa, it would be quite difficult for a cell to successfully incorporate all of the genes at once, losing other pre-existing genes as necessary, to form a functional outer membrane (Antunes et al. 2016). It would be much easier for a cell to lose an outer membrane (or other complex structure in general) than to gain one. For example, this could possibly occur if a bacterium had a mutation that resulted in a thicker cell wall, which in turn could affect the anchoring of the outer membrane to the cell wall (Cavalier-Smith 2006), or alternatively, mutations could occur in a gene for an anchoring protein, resulting in a defective protein and the destabilization and detachment of the outer membrane (Antunes et al. 2016).

To summarize: over the course of the evolution of bacteria, it is apparent that in multiple instances monoderm cells evolved from diderm ancestors (at least in the case of the Firmicutes). But we still don't know with any certainty whether the ancestor shared by all bacteria was diderm or monoderm.

In all likelihood, the first cells to exist on Earth were monoderm. Monoderms have a simpler cell envelope, and evolution, over the long term, tends to build more complex structures from simpler pre-existing designs. As noted by Canadian researcher Radhey Gupta (Gupta 2011), it is difficult to imagine any simple model where a cell with two membranes could have evolved without the initial development of a cell with a single membrane.

How could a monoderm cell become diderm?

Thus, it is likely that at some time in the past diderm cells evolved from a monoderm ancestor. This transition is not as simple as losing an outer membrane. Besides acquiring all the genes needed for an outer membrane, the cell would need to undergo some process to form that membrane. Cell membranes aren't synthesized from scratch— they're inherited (Beisson 2008). So it doesn't matter if all the genes are present to build a membrane. There has to be some pre-existing membrane structure, which then can be modified and adapted as necessary from the gene products that are produced by the cell.

So, as far as we know, the outer membrane must have been formed from some other membrane. But how? Thanks to research performed by Elitza Tocheva and colleagues (Tocheva et al. 2011) we know that outer membranes can be formed from cytoplasmic membranes as the result of a process known as sporulation (or more specifically endospore formation).

Some bacteria are able to produce spores when growth conditions become unfavorable. Spores are durable dormant cells that are capable of surviving harsh conditions that would kill normal, biologically active cells. Once environmental conditions become favorable again, a spore "germinates" and a biologically active cell grows out from it.

Endospores are the type of spores produced by bacteria from the phylum Firmicutes. They are the most durable cells known on earth— being capable of surviving heat, UV and gamma radiation, desiccation, and oxidizing agents (Nicholson et al. 2000). Endospores can successfully germinate after remaining dormant for extremely long periods of time— sometimes thousands of years or longer (Gest and Mandelstam 1987, Kennedy et al. 1994, Potts 1994).

Spore formation occurs primarily in monoderm bacteria, although some diderm species are known to form spores. Endospores of monoderm and diderm bacteria have the same structure and they are formed in the same manner (Tocheva et al. 2016). Sporulation appears to have evolved from a cell replication event that went awry (Tocheva et al. 2011). It starts off like cell replication, with the bacterium replicating and segregating its DNA, and forming a septum between the two halves of the cell. However, instead of dividing into two daughter cells, the membrane of one half (referred to as the mother cell) begins growing around the other half (the soon to be spore), eventually surrounding it entirely. The maturing spore, located within the mother cell, is enclosed by two membranes, both of which were derived from the cytoplasmic membrane of the original mother cell. Upon maturation of the spore, the mother cell breaks open, releasing the spore (Figure 11).

When the spore of a monoderm bacterium germinates, the outer membrane of the spore is lost and the inner membrane of the spore becomes the cytoplasmic membrane of the outgrowing cell. However, in a diderm bacterium, both of the spore's membranes are retained and they become the cytoplasmic and outer membranes of the emerging cell (Tocheva et al. 2011).

Tocheva et al. 2011 examined sporulation in the diderm Acetonema longum. Through electron cryotomography imaging, they observed that the spore of this bacterium was surrounded by two membranes that were derived from the cytoplasmic membrane of the mother cell. They also found that following germination, both membranes of the spore were retained to become the cytoplasmic and outer membranes of the outgrowing cell.^[13] Acetonema longum has a classical gram-negative cell envelope, with an outer membrane containing LPS and typical outer membrane proteins. Therefore, at some point in the sporulation/germination process, the outer cytoplasmic membrane of the spore was transformed into a bona fide outer membrane.

Since the spores of both monoderm and diderm bacteria are surrounded by two membranes and have the same overall structure, it's easy to image a scenario where an ancient sporulating monoderm could have given rise to a diderm. There would simply need to be some mutation(s) causing the outer membrane of the spore to be retained, so that the outgrowing cell would have an outer membrane. Likewise a monoderm species could possibly evolve from a sporulating diderm if a mutation occurred that resulted in the loss of the outer spore membrane.

The first diderms to evolve likely had an outer membrane that was very similar in composition to the cytoplasmic membrane. But over time, diderm bacteria would have acquired genes that enhanced the function of the outer membrane, leading to the present day outer membranes (Tocheva et al. 2011). There are at least two unique types of outer membranes among the known bacteria— the LPS outer membrane (characteristic of most diderm bacteria), and the mycolic acid-based outer membrane (found in some bacteria from the Phylum Actinobacteriota). Thus it is possible that cells with outer membranes evolved from monoderm progenitors on at least two occasions (Sutcliffe and Dover 2016).

The Cell Wall and Osmosis

A bacterium's cell wall is a porous protective barrier that helps to maintain the shape of the cell and prevent the cell from bursting (lysing) in hypotonic environments. The cell wall surrounds the semi-permeable cytoplasmic membrane, which encloses the contents of the cell. Water flows freely across the membrane, but the membrane does not allow the free passage of many dissolved particles. Because the solute (particle) concentration inside a cell is typically higher than that of the surrounding medium, there is a tendency for water to flow (via osmosis) from the environment into the cell. Water will continue to flow into the cell until the solute concentration is the same on both sides of the membrane or until the force or pressure of water flowing into the cell is counterbalanced by the force or pressure inside the cell. Without a cell wall or other reinforcement of the cell membrane, the influx of water into a cell typically causes the cell to swell, stretching and stressing the membrane until it ruptures and the cell lyses. However, when a membrane is reinforced by a cell wall, the cell wall prevents the membrane from expanding when water enters the cell. Instead, pressure builds up inside the cell from the inflow of water, but the cell wall is strong enough to resist the pressure and the cell is able to hold its shape. Because of the need to counterbalance the force of osmosis, the pressure inside some gram-positive bacterial cells may be as high as 20 atmospheres or 300 pounds per square inch (Prescott et al. 1993, Mitchell and Moyle 1956). Gram-negative bacteria, because of their thinner cell walls, are believed to support much lower internal pressures (Koch and Doyle 1986) and thus lower internal solute concentrations. Besides tolerating a range of internal pressures, bacteria can also manage osmotic stresses by accumulating or releasing solute particles (Wood 2015).

Groups of Bacteria

For asexual organisms, such as bacteria, the classical definition of a species— a group of individuals that interbreed and produce fertile offspring— does not apply. In order for a scientist to determine whether a bacterium belongs to a particular species (or any other taxonomic group), he or she has to utilize arbitrary criteria to establish cut-off points between the different groups.

Currently there is no standard definition for what constitutes a species of bacterium. The assignment of bacterial species to higher taxonomic ranks (e.g., genus, family, etc.) is also problematic and lacks a consistent, standardized approach. Bacteria have been classified based on phenotypic characteristics, genomic characteristics, or a combination of the two. Some of the methods that have been used to classify bacteria are listed below.

• Variation in DNA base composition (mol% G+C content) (Fournier et al. 2006).
• DNA-DNA hybridization. (Wayne et al. 1987, Konstantinidis et al. 2006).
• Gene sequence similarity using select DNA markers (including 16S rRNA) (Chan et al. 2012, Varghese et al. 2015)
• Whole-genome sequencing (Church et al. 2020)
• Presence of certain metabolites, morphological features, growth requirements, or other biochemical or enzymological characters (Varghese et al. 2015, Chan et al. 2012).

Compared to macroscopic organisms, bacteria are very challenging to study and classify. They're generally invisible to the unaided eye, they're found in all sorts of habitats, and the vast majority of them can't be successfully isolated and cultured. To obtain an idea of the total biodiversity of bacteria on Earth, researchers need to sample all imaginable habitats and all types of environmental media (e.g., soils, waters, tissues, etc.). Fortunately, with recent advances in molecular biology, scientists can now identify genomes of bacteria from environmental samples— without having to culture the organisms. So now, new taxa of bacteria are being identified at a rapid rate.

Bacterial taxa that have no cultured representatives (i.e., no member taxa that have been isolated and grown in pure culture) are referred to as "candidate" taxa and are written with the Latin word candidatus preceeding the taxon name (e.g., candidate phylum [or division] Zixibacteria, also referred to as Candidatus Zixibacteria, or abbreviated Ca. Zixibacteria). Note when Candidatus is used with a species name, the word Candidatus is italicized but the species name is not, e.g., Candidatus Scalindua brodae. Currently there are more candidate phyla of bacteria than there are bacterial phyla with cultured representatives.

The table below provides information on various phyla of bacteria. The list shown is a subset of the phyla identified in the Genome Taxonomy Database (GTDB 2018). Whereas the GTDB contains all identified phyla, both cultured and uncultured, the list below includes only those phyla that have at least one cultured representative. One candidate phylum— Entotheonellota— is included in the list below. Although Entotheonellota contains a species that has been cultured, that species (a symbiont) has not been grown in pure culture (only in mixed culture with its host), and therefore is considered to have candidate status.

The classification of bacteria is a work in progress. Hopefully over time, classification methodologies will become more standardized and universally applied. It is anticipated that many, many more taxa of bacteria will be discovered and described in the future, and the taxonomic placement of known bacteria will change somewhat as more data become available and as phylogenetic methods are improved.