Gram-negative bacteria are bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counterstain (commonly safranin) is added after the crystal violet, coloring all gram-negative bacteria with a red or pink color. The counterstain is used to visualize the otherwise colorless gram-negative bacteria whose much thinner peptidoglycan layer does not retain crystal violet. The test itself is useful in classifying two distinct types of bacteria based on the structural differences of their bacterial cell walls. Gram-positive bacteria retain the crystal violet dye when washed in a decolorizing solution. Compared with gram-positive bacteria, gram-negative bacteria are more resistant against antibiotics, despite their thinner peptidoglycan layer, because of their additional, relatively impermeable lipid membrane.
The pathogenic capability of gram-negative bacteria is often associated with certain components of gram-negative cell envelope, in particular, the lipopolysaccharide layer (also known as LPS or endotoxin layer). In humans, LPS triggers an innate immune response characterized by cytokine production and immune system activation. Inflammation is a common result of cytokine (from the Greek cyto, cell and kinesis, movement) production, which can also produce host toxicity. The innate immune response to LPS, however, is not synonymous with pathogenicity, or the ability to cause disease. In fact, the innate immune response is triggered by LPS alone, isolated from bacteria.
The following characteristics are displayed for gram-negative bacteria:
- Cytoplasmic membrane
- Thin peptidoglycan layer (which is much thicker in gram-positive bacteria)
- Outer membrane containing lipopolysaccharide (LPS, which consists of lipid A, core polysaccharide, and O antigen) in its outer leaflet and phospholipids in the inner leaflet
- Porins exist in the outer membrane, which act like pores for particular molecules
- There is a space between the layers of peptidoglycan and the secondary cell membrane called the periplasmic space
- The S-layer is directly attached to the outer membrane, rather than the peptidoglycan
- If present, flagella have four supporting rings instead of two
- No teichoic acids or lipoteichoic acids are present
- Lipoproteins are attached to the polysaccharide backbone.
- Most of them contain Braun's lipoprotein, which serves as a link between the outer membrane and the peptidoglycan chain by a covalent bond
- Most do not sporulate (Coxiella burnetii, which produces spore-like structures, is a notable exception)
Along with cell shape, Gram staining is a rapid diagnostic tool and once was used to group species at the subdivision of Bacteria.
In fact, historically the kingdom Monera was divided into four divisions based on Gram staining: Firmacutes (+), Gracillicutes (−), Mollicutes (0) and Mendocutes (var.). Since 1987, the monophyly of the gram-negative bacteria has been disproven with molecular studies. However some authors, such as Cavalier-Smith still treat them as a monophyletic clade and refer to the group as subkingdom "Negibacteria".
Outer cell membrane bacterial classification
It is important to point out that though the bacteria are traditionally divided into two main groups, gram-positive and gram-negative, based on their Gram-stain retention, this classification system is ambiguous, as it can refer 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 phylogenetic character as these two kinds of bacteria do not form phylogenetically coherent groups. However, 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 two kinds of cells are distinguished from each other based upon the presence or absence of an outer lipid membrane, which is a reliable and fundamental characteristic of bacterial cells. All gram-positive bacteria are bounded by only 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 that are bounded by a single membrane, but stain gram-negative due to either lack of the peptidoglycan layer (viz., mycoplasmas) or their inability to retain the gram-stain because of 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 both a cytoplasmic membrane as well as an outer cell membrane and they contain only a thin layer of peptidoglycan (2–3 nm) in between these two membranes. The presence of both 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 also 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 different 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) evolved 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, but 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.
Additionally, a number of bacterial taxa (viz. Negativicutes, Fusobacteria, Synergistetes and Elusimicrobia) that are either part of the phylum Firmicutes or branches in its proximity are also 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) from these other atypical diderm bacteria as well as other phyla of monoderm bacteria (e.g. Actinobacteria, Firmicutes, Thermotogae, Chloroflexi). 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.
The proteobacteria are a major group of gram-negative bacteria, including Escherichia coli (E. coli), Salmonella, Shigella, and other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella and numerous others. Other notable groups of gram-negative bacteria include the cyanobacteria, spirochaetes, green sulfur and green non-sulfur bacteria.
Medically relevant gram-negative cocci include the three organisms, which cause a sexually transmitted disease (Neisseria gonorrhoeae), a meningitis (Neisseria meningitidis), and respiratory symptoms (Moraxella catarrhalis).
Medically relevant gram-negative bacilli include a multitude of species. Some of them primarily cause respiratory problems (Hemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), primarily urinary problems (Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens), and primarily gastrointestinal problems (Helicobacter pylori, Salmonella enteritidis, Salmonella typhi).
Gram-negative bacteria associated with nosocomial infections include Acinetobacter baumannii, which cause bacteremia, secondary meningitis, and ventilator-associated pneumonia in intensive-care units of hospital establishments.
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One of the several unique characteristics of gram-negative bacteria is the structure of the outer membrane. The outer leaflet of the membrane comprises a complex lipopolysaccharide whose lipid portion acts as an endotoxin. If endotoxin enters the circulatory system, it causes a toxic reaction, with the sufferer developing a high temperature, high respiration rate, and low blood pressure. This may lead to endotoxic shock, which may be fatal.
This outer membrane protects the bacteria from several antibiotics, dyes, and detergents that would normally damage the inner membrane or cell wall (peptidoglycan). The outer membrane provides these bacteria with resistance to lysozyme and penicillin. However, alternative medicinal treatments such as lysozyme with EDTA and the antibiotic ampicillin have been developed to combat the protective outer membrane of some pathogenic gram-negative organisms. Other drugs can be used, significant ones being chloramphenicol, streptomycin, and nalidixic acid.
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