Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counterstain (safranin or fuchsine) and appearing red or pink. Gram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall. Gram-positive cell walls typically lack the outer membrane found in Gram-negative bacteria.
The following characteristics are generally present in a Gram-positive bacterium:
- cytoplasmic lipid membrane
- thick peptidoglycan layer
- capsule polysaccharides (only in some species)
- flagellum (only in some species)
- if present, it contains two rings for support as opposed to four in Gram-negative bacteria because Gram-positive bacteria have only one membrane layer.
- The individual peptidoglycan molecules are cross-linked by pentaglycine chains by a DD-transpeptidase enzyme. In gram-negative bacteria, the transpeptidase creates a covalent bond directly between peptidoglycan molecules, with no intervening bridge.
Both Gram-positive and Gram-negative bacteria may have a membrane called an S-layer. In Gram-negative bacteria, the S-layer is attached directly to the outer membrane. In Gram-positive bacteria, the S-layer is attached to the peptidoglycan layer. Unique to Gram-positive bacteria is the presence of teichoic acids in the cell wall. Some particular teichoic acids, lipoteichoic acids, have a lipid component and can assist in anchoring peptidoglycan, as the lipid component is embedded in the membrane.
Along with cell shape, Gram staining is a rapid diagnostic tool of use to group species of bacteria. In traditional and even some areas of contemporary microbiological practice, such staining, alongside growth requirement and antibiotic susceptibility testing, and other macroscopic and physiologic tests, forms the full basis for classification and subdivision of the bacteria (e.g., see figure and pre-1990 versions of Bergey's Manual).
As such, historically, the kingdom Monera was divided into four divisions based primarily on Gram staining: Firmicutes (positive in staining), Gracillicutes (negative in staining), Mollicutes (neutral in staining) and Mendocutes (variable in staining). 16S ribosomal RNA phylogenetic studies of Carl Woese (Department of Microbiology, University of Illinois) and collaborators and colleagues, the monophyly of the Gram-positive bacteria has been challenged, with striking productive implications for the therapeutic and general study of these organisms. Based on molecular studies of 16S sequences, Woese recognised twelve bacterial phyla, two being Gram-positive: high-GC Gram-positives and low-GC Gram-positives (where G and C refer to the guanine and cytosine content in their genomes), which are now referred to by these names, or as Actinobacteria and Firmicutes. The former, the Actinobacteria, are the high GC content Gram-positive bacteria and contains genera such as Corynebacterium, Mycobacterium, Nocardia and Streptomyces. The latter, the Firmicutes are the "low-GC" Gram-positive bacteria, which actually have 45%–60% GC content but lower than that of the Actinobacteria.
Importance of the outer cell membrane in bacterial classification 
Although the bacteria are traditionally divided into two main groups, Gram-positive and Gram-negative, based upon their Gram-stain retention property, this classification system is ambiguous as it refers to three distinct aspects (staining result, cell-envelope organization, taxonomic group), which do not necessarily coalesce for some bacterial species. The Gram-positive and Gram-negative staining response is also not a reliable characteristic as these two kinds of bacteria do not form phylogenetic coherent groups. However, although Gram-staining response of bacteria is an empirical criterion, its basis lies in the marked differences in the ultrastructure and chemical composition of the two main kinds of prokaryotic cells that are found in nature. These kinds of cells are distinguished from each other based upon the presence or absence of an outer lipid membrane, which is a more reliable and fundamental characteristic of the bacterial cells.
All Gram-positive bacteria are bounded by a single unit lipid membrane, and they generally contain a thick layer (20-80 nm) of peptidoglycan responsible for retaining the Gram-stain. A number of other bacteria which are bounded by a single membrane, but which stain Gram-negative due to either lack of the peptidoglycan layer (viz., mycoplasmas) or their inability to retain the Gram-stain due to their cell wall composition, also show close relationship to the gram-positive bacteria. For the bacterial (prokaryotic) cells that are bounded by a single cell membrane the term "monoderm bacteria" or "monoderm prokaryotes" has been proposed.
In contrast to Gram-positive bacteria, all archetypical Gram-negative bacteria are bounded by a cytoplasmic membrane and an outer cell membrane; they contain only a thin layer of peptidoglycan (2-3 nm) between these membranes. The presence of inner and outer cell membranes defines a new compartment in these cells: the periplasmic space or the periplasmic compartment. These bacteria/prokaryotes have been designated as "diderm bacteria." The distinction between the monoderm and diderm prokaryotes is supported by conserved signature indels in a number of important proteins (viz. DnaK, GroEL). Of these two structurally distinct groups of prokaryotic organisms, monoderm prokaryotes are indicated to be ancestral. Based upon a number of observations including that the Gram-positive bacteria are the major producers of antibiotics and that Gram-negative bacteria are generally resistant to them, it has been proposed that the outer cell membrane in Gram negative bacteria (diderms) has as a protective mechanism against antibiotic selection pressure. Some bacteria, such as Deinococcus, which stain Gram-positive due to the presence of a thick peptidoglycan layer and also possess an outer cell membrane are suggested as intermediates in the transition between monoderm (Gram positive) and diderm (Gram-negative) bacteria. The diderm bacteria can also be further differentiated between simple diderms lacking lipopolysaccharide, the archetypical diderm bacteria where the outer cell membrane contains lipopolysaccharide and the diderm bacteria where outer cell membrane is made up of mycolic acid.
In general, Gram-positive bacteria have a single lipid bilayer (monoderms), whereas Gram-negative have two (diderms). Some taxa lack peptidoglycan (such as the domain Archaea, the class Mollicutes, some members of the Rhickettsiales, and the insect-endosymbionts of the Enterobacteriales) and are Gram-variable. This, however, does not always hold true. The Deinococcus-Thermus bacteria have Gram-positive stains, although they are structurally similar to Gram-negative bacteria with two layers (diderms). The Chloroflexi have a single layer, yet (with some exceptions) stain negative. Two related phyla to the Chloroflexi, the TM7 clade and the Ktedonobacteria, are also monoderms.
Some Firmicute species are not Gram-positive; these belong to the class Mollicutes (alternatively considered a class of the phylum Tenericutes), which lack peptidoglycan (Gram-indeterminate), and the class Negativicutes, which includes Selenomonas and which stain Gram-negative. Additionally, a number of bacterial taxa (viz. Negativicutes, Fusobacteria, Synergistetes and Elusimicrobia) that are either part of the phylum Firmicutes or branch in its proximity are found to possess a diderm cell structure. However, a conserved signature indel (CSI) in the HSP60 (GroEL) protein distinguishes all traditional phyla of Gram-negative bacteria (e.g., Proteobacteria, Aquificae, Chlamydiae, Bacteroidetes, Chlorobi, Cyanobacteria, Fibrobacteres, Verrucomicrobia, Planctomycetes, Spirochetes, Acidobacteria, etc.) from these other atypical diderm bacteria as well as other phyla of monoderm bacteria (e.g., Actinobacteria, Firmicutes, Thermotogae, Chloroflexi, etc.). The presence of this CSI in all sequenced species of conventional LPS-containing Gram-negative bacterial phyla provides evidence that these phyla of bacteria form a monophyletic clade and that no loss of the outer membrane from any species from this group has occurred.
Most pathogens in humans are Gram-positive organisms. In the classical sense, six Gram-positive genera are typically pathogenic in humans. Two of these, Streptococcus and Staphylococcus, are cocci (sphere-shaped bacteria). The remaining organisms are bacilli (rod-shaped bacteria) and can be subdivided based on their ability to form spores. The non-spore formers are Corynebacterium and Listeria (a coccobacillus), whereas Bacillus and Clostridium produce spores. The spore-forming bacteria can again be divided based on their respiration: Bacillus is a facultative anaerobe, while Clostridium is an obligate anaerobe.
See also 
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- 3D structures of proteins associated with plasma membrane of Gram-positive bacteria
- 3D structures of proteins associated with outer membrane of Gram-positive bacteria
- Gram Staining Procedure and Images