Gram-negative bacteria are a group of bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation, making positive identification possible. The thin peptidoglycan layer of their cell wall is sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane. After staining with crystal violet, an alcohol wash is applied which decolorizes the bacteria showing that their peptidoglycan layer is too thin to retain the stain and enabling identification. A counterstain (safranin or fuchsine) is then added which recolorizes the bacteria red or pink.
Gram-positive bacteria have a thicker peptidoglycan layer in their cell wall outside the cell membrane, which retains the crystal violet stain during the alcohol wash, so long as it is timed correctly. The counter stain may also be absorbed by gram-positive bacteria but the darker crystal violet stain predominates.
Gram-negative bacteria display the following characteristics:
- An inner cell membrane is present (cytoplasmic)
- A thin peptidoglycan layer is present (This is much thicker in gram-positive bacteria)
- Has outer membrane containing lipopolysaccharides (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
- Between the outer membrane and the cytoplasmic membrane there is a space filled with a concentrated gel-like substance called periplasm
- The S-layer is directly attached to the outer membrane rather than to the peptidoglycan
- If present, flagella have four supporting rings instead of two
- Teichoic acids or lipoteichoic acids are absent
- Lipoproteins are attached to the polysaccharide backbone
- Some contain Braun's lipoprotein, which serves as a link between the outer membrane and the peptidoglycan chain by a covalent bond
- Most, with very few exceptions, do not form spores
Along with cell shape, Gram staining is a rapid diagnostic tool and once was used to group species at the subdivision of Bacteria. 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
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Though bacteria are traditionally divided into the two main groups of gram-positive and gram-negative, based on their Gram stain retention, this classification system can be ambiguous; the results of Gram-staining, the cell-envelope organization of the bacteria, and the taxonomic group that the bacteria belongs to, do not necessarily match up for some bacterial species. A positive or negative gram stain is also not a reliable characteristic for phylogenetic sorting, as these gram-positive and gram-negative bacteria do not form phylogenetically coherent groups. However, Gram staining response of bacteria is an empirical criterion; it is based on 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.
The term Monoderm bacteria or Monoderm prokaryotes has been proposed for the bacterial (prokaryotic) cells that are bounded by a single cell membrane. All gram-positive bacteria are bounded by only a single unit lipid membrane and generally contain a thick layer (20–80 nm) of peptidoglycan, which is responsible for retaining the Gram stain. A number of other bacteria that are bounded by a single membrane, but stain gram-negative either because they lack the peptidoglycan layer (viz., mycoplasmas) or are unable to retain the Gram stain because of their cell wall composition, also show a close evolutionary relationship to the gram-positive bacteria.
The term Diderm Bacteria has been proposed for bacteria/prokaryotes that are bounded by a cytoplasmic membrane and an outer cell membrane. In contrast to gram-positive bacteria, all archetypical gram-negative bacteria have a cytoplasmic membrane as well as and an outer cell membrane, and contain only a thin layer of peptidoglycan (2–3 nm) between the membranes. The inner and outer cell membranes form a new compartment in these cells, the periplasmic space or the periplasmic compartment. 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 reactors to antibiotics and that gram-negative bacteria are, in general, 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, in which the outer cell membrane contains lipopolysaccharide, and the diderm bacteria, in which outer cell membrane is made up of mycolic acid.
In addition, 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 lipopolysaccharide-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 etc. 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 four organisms that cause a sexually transmitted disease (Neisseria gonorrhoeae), a meningitis (Neisseria meningitidis), and respiratory symptoms (Moraxella catarrhalis, Haemophilus Influenzae).
Medically relevant gram-negative bacilli include a multitude of species. Some of them cause primarily respiratory problems (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 hospital-acquired infections include Acinetobacter baumannii, which cause bacteremia, secondary meningitis, and ventilator-associated pneumonia in hospital intensive-care units.
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One of the several unique characteristics of gram-negative bacteria is the structure of the bacterial outer membrane. The outer leaflet of this membrane comprises a complex lipopolysaccharide (LPS) whose lipid portion acts as an endotoxin. If gram-negative bacteria enter the circulatory system, the liposaccharide can cause a toxic reaction. This results in fever, an increased respiratory rate, and low blood pressure. This may lead to life-threatening condition of endotoxic shock.
The outer membrane protects the bacteria from several antibiotics, dyes, and detergents that would normally damage either the inner membrane or the cell wall (made of 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 also be used, significant ones being chloramphenicol, streptomycin, and nalidixic acid. Chloramphenicol is rarely used in the EU due to the association with drug induced pancytopenia.
The pathogenic capability of gram-negative bacteria is often associated with certain components of their membrane, in particular, the LPS. In humans, the presence of LPS triggers an innate immune response, activating the immune system and producing cytokines (hormonal regulators). Inflammation is a common reaction to cytokine 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.
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