Gram-positive bacteriaGram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is incontrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking upthe counterstain (safranin or fuchsine) and appearing red or pink. Gram-positive organisms are ableto 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. ClassificationThe following characteristics are generally present in a Gram-positive bacterium: 1. cytoplasmic lipid membrane 2. thick peptidoglycan layer o teichoic acids and lipoids are present, forming lipoteichoic acids, which serve to act as chelating agents, and also for certain types of adherence. 3. capsule polysaccharides (only in some species) 4. flagellum (only in some species) o 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. 5. 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. InGram-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-positivebacteria 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 thelipid component is embedded in the membrane.ClassificationAlong with cell shape, Gram staining is a rapid diagnostic tool of use to group species ofBacteria. In traditional and even some areas of contemporary microbiological practice, suchstaining, alongside growth requirement and antibiotic susceptibility testing, and othermacroscopic and physiologic tests, forms the full basis for classification and subdivision ofthe Bacteria (e.g., see Figure, and pre-1990 versions of Bergeys Manual).
Species identification hierarchy in clinical settings.As such, historically, the kingdom Monera was divided into four divisions based primarily onGram staining: Firmicutes (positive in staining), Gracillicutes (negative in staining),Mollicutes (neutral in staining) and Mendocutes (variable in staining).Since 1987 and the seminal 16S ribosomal RNA phylogenetic studies of Carl Woese(Department of Microbiology, University of Illinois) and collaborators and colleagues, themonophyly of the Gram-positive bacteria has been challenged, with striking productiveimplications for the therapeutic and general study of these organisms. Based on molecularstudies 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 theguanine 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 contentGram-positive bacteria and contains genera such as Corynebacterium, Mycobacterium,Nocardia and Streptomyces. The latter, the Firmicutes are the "low-GC" Gram-positivebacteria, which actually have 45%–60% GC content but lower than that of theActinobacteria,. The Firmicutes contain the well-known genera that are majority of Gram-positives of medical interest: Staphylococcus, Streptococcus, Enterococcus (cocci), Bacillus,Clostridium and Listeria (bacilli/rods). This group also been expanded to include theMycoplasma, or Mollicutes, bacteria-like organisms that lack cell walls and cannot be Gram-stained, but appear to have derived evolutionarily from such forms.
Despite the wide acceptance and practical record of utility of the new molecular phylogeny, asmall group, including Cavalier-Smith, still treat the Monera as a monophyletic clade andrefer to the group as division "Posibacteria".Importance of the Outer Cell Membrane in Bacterial ClassificationIt is important to point out that although the bacteria are traditionally divided into two maingroups, 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 somebacterial species. The Gram-positive and Gram-negative staining response is also nota reliable characteristic as these two kinds of bacteria do not form phylogenetic coherentgroups. However, although Gram-staining response of bacteria is an empirical criterion, itsbasis lies in the marked differences in the ultrastructure and chemical composition of the twomain kinds of prokaryotic cells that are found in nature. These two kinds of cells aredistinguished from each other based upon the presence or absence of an outer lipidmembrane, which is a more reliable and fundamental characteristic of the bacterial cells.All Gram-positive bacteria are bounded by only a single unit lipid membrane and theygenerally contain a thick layer (20-80 nm) of peptidoglycan responsible for retaining theGram-stain. A number of other bacteria which are bounded by a single membrane, but whichstain Gram-negative due to either lack of the peptidoglycan layer (viz., mycoplasmas) or theirinability to retain the Gram-stain due to their cell wall composition, also show closerelationship to the gram-positive bacteria. For the bacterial (prokaryotic) cells that arebounded by a single cell membrane the term Monoderm Bacteria or MonodermProkaryotes has been proposed. In contrast to Gram-positive bacteria, all archetypicalGram-negative bacteria, are bounded by both a cytoplasmic membrane as well as an outercell membrane and they contain only a thin layer of peptidoglycan (2-3 nm) in between thesetwo membranes. The presence of both inner and outer cell membranes defines a newcompartment in these cells, the periplasmic space or the periplasmic compartment. Thesebacteria/prokaryotes have been designated as Diderm Bacteria. The distinctionbetween the monoderm and diderm prokaryotes is also supported by conserved signatureindels in a number of important proteins (viz. DnaK, GroEL). Of these twostructurally distinct groups of prokaryotic organisms, monoderm prokaryotes are indicated tobe ancestral. Based upon a number of different observations including that the Gram-positivebacteria are the major producers of antibiotics and that Gram-negative bacteria are generallyresistant 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. Somebacteria such as Deinococcus, which stain Gram-positive due to the presence of a thickpeptidoglycan layer, but also possess an outer cell membrane are suggested as intermediatesin the transition between monoderm (Gram positive) and diderm (Gram-negative)bacteria. The diderm bacteria can also be further differentiated between simple didermslacking lipopolysaccharide, the archetypical diderm bacteria where the outer cell membranecontains lipopolysaccharide and the diderm bacteria where outer cell membrane is made upof mycolic acid.ExceptionsIn 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, theclass Mollicutes, some members of the Rhickettsiales, and the insect-endosymbionts of the
Enterobacteriales) and are Gram-variable. This, however, does not always hold true. TheDeinococcus-Thermus bacteria have Gram-positive stains, although they are structurallysimilar to Gram-negative bacteria with two layers (diderms). The Chloroflexi have a singlelayer, 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 whichstain Gram-negative. Additionally, a number of bacterial taxa (viz. Negativicutes,Fusobacteria, Synergistetes and Elusimicrobia) that are either part of the phylum Firmicutesor branch 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 traditionalphyla 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 ofmonoderm bacteria (e.g. Actinobacteria, Firmicutes, Thermotogae, Chloroflexi, etc.). Thepresence of this CSI in all sequenced species of conventional LPS-containing Gram-negativebacterial phyla provides evidence that these phyla of bacteria form a monophyletic clade andthat no loss of the outer membrane from any species from this group has occurred.PathogenesisMost pathogens in humans are Gram-positive organisms. In the classical sense, six Gram-positive genera are typically pathogenic in humans. Two of these, Streptococcus andStaphylococcus, 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-sporeformers are Corynebacterium and Listeria (a coccobacillus), whereas Bacillus andClostridium produce spores. The spore-forming bacteria can again be divided based ontheir respiration: Bacillus is a facultative anaerobe, while Clostridium is an obligate anaerobe.