Gram-negative bacteria
Gram-negative bacteria are a diverse group of prokaryotic microorganisms characterized by their unique cell wall structure, which includes a thin peptidoglycan layer located in the periplasmic space between an inner cytoplasmic membrane and an outer membrane composed primarily of lipopolysaccharides (LPS).[1] They are distinguished from Gram-positive bacteria through the Gram staining procedure, where they fail to retain the crystal violet-iodine complex during decolorization with acetone-alcohol, resulting in a pink or red appearance after counterstaining with safranin or fuchsin due to the lipid-rich outer membrane and low peptidoglycan content.[2] This classification, developed by Hans Christian Gram in 1884, reflects fundamental differences in cell envelope architecture that influence antibiotic susceptibility, pathogenicity, and environmental adaptability.[2][3] The cell envelope of Gram-negative bacteria provides a selective permeability barrier, with the outer membrane's LPS—consisting of lipid A, a core polysaccharide, and an O-antigen chain—playing a critical role in protecting against hydrophobic compounds, detergents, and many antibiotics while also serving as an endotoxin that can elicit strong inflammatory responses in hosts.[1] The thin peptidoglycan layer, only a few nanometers thick, offers structural rigidity and shape maintenance (typically rod-shaped bacilli), but lacks the thickness seen in Gram-positive counterparts, allowing easier access for certain lytic enzymes.[1] These bacteria are ubiquitous in nature, thriving in diverse habitats such as soil, freshwater, and the gastrointestinal tracts of animals, where they perform essential ecological roles like nutrient cycling.[4] In clinical contexts, Gram-negative bacteria are major opportunistic pathogens, particularly in healthcare settings, causing severe infections including pneumonia, urinary tract infections, bloodstream bacteremia, wound and surgical site infections, and meningitis, often in immunocompromised patients or those with invasive devices.[5] Notable genera include Escherichia (e.g., E. coli), Pseudomonas (e.g., P. aeruginosa), Salmonella, Klebsiella, and Neisseria, which account for a significant portion of nosocomial infections and exhibit high morbidity and mortality rates due to their ability to form biofilms and evade host defenses.[4] Their intrinsic and acquired resistance mechanisms—such as porin mutations, efflux pumps, and beta-lactamase production—render them challenging to treat, with some strains resistant to nearly all available antibiotics, underscoring the urgent need for new therapeutic strategies and infection prevention measures like hand hygiene and antibiotic stewardship.[5][4]Characteristics
Gram Staining and Morphology
Gram-negative bacteria are defined by their response to the Gram staining technique, a differential method that distinguishes them from Gram-positive bacteria based on cell wall properties. Developed by Danish bacteriologist Hans Christian Gram in 1884 while examining lung tissue from pneumonia patients, the procedure involves applying crystal violet dye, followed by iodine mordant, alcohol decolorization, and a safranin counterstain.[6] In Gram-negative bacteria, the thin peptidoglycan layer in the cell wall fails to retain the crystal violet-iodine complex during alcohol treatment, allowing the dye to wash out and the cells to take up the pink safranin counterstain, appearing red or pink under a microscope.[2][7] Morphologically, Gram-negative bacteria exhibit diverse shapes, though they commonly appear as bacilli (rods), cocci (spheres), or spirilla (rigid spirals). Rod-shaped forms, such as Escherichia coli, are prevalent in genera like Escherichia and Pseudomonas, often occurring singly or in chains.[8] Cocci are seen in pairs or clusters, exemplified by Neisseria gonorrhoeae, a diplococcus responsible for gonorrhea, with cells typically flattened on adjacent sides.[9] Spirilla, like Spirillum minus associated with rat-bite fever, feature a helical or coiled structure that aids in identification.[10] These shapes are observed via light microscopy after Gram staining, providing initial clues for bacterial identification.[11] In terms of size, Gram-negative rods generally measure 0.5–1.0 μm in width and 1.0–5.0 μm in length, as illustrated by E. coli cells averaging 1.0–2.0 μm long and 1.0 μm wide.[12] Cocci, such as those in Neisseria species, range from 0.6–1.0 μm in diameter, while spirilla can extend 3–5 μm or more in length depending on coil turns.[9] These dimensions contribute to their visibility under standard magnification and influence their ecological roles, though variations occur across species.[11]Cell Envelope Structure
The cell envelope of Gram-negative bacteria consists of a tripartite structure comprising an inner cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane, which collectively provide mechanical strength and act as a permeability barrier. The inner cytoplasmic membrane is a symmetric phospholipid bilayer approximately 7 nm thick, primarily composed of phospholipids such as phosphatidylethanolamine and phosphatidylglycerol, serving as the site for energy transduction and active transport. The peptidoglycan layer, situated in the periplasm, is notably thin at 2–7 nm and typically consists of 1–2 layers of cross-linked N-acetylglucosamine and N-acetylmuramic acid disaccharides, offering rigidity while allowing flexibility for cell growth. The outer membrane forms an asymmetric lipid bilayer about 7–8 nm thick, with phospholipids in the inner leaflet and lipopolysaccharides (LPS) dominating the outer leaflet, which contributes to the envelope's selective permeability and protection against environmental stresses.[1] Between the inner and outer membranes lies the periplasmic space, a gel-like aqueous compartment approximately 10–20 nm wide that is more viscous than the cytoplasm due to its high protein density and peptidoglycan matrix. This region houses a diverse array of enzymes, including proteases, nucleases, and binding proteins, which facilitate nutrient acquisition and processing, such as the breakdown of imported substrates for uptake into the cytoplasm. Additionally, the periplasm contains stress response machinery, including chaperones and quality control proteins like DegP, that protect against protein misfolding and envelope damage, ensuring cellular homeostasis under varying conditions.[1][13] The outer membrane's asymmetry is maintained by LPS molecules, which anchor deeply into the bilayer and interact via divalent cations like Mg²⁺ to form a tightly packed, low-fluidity surface. LPS is structured in three domains: lipid A, a phosphorylated glucosamine disaccharide with 6–7 saturated acyl chains serving as the hydrophobic anchor and endotoxic component; a core oligosaccharide linking lipid A to the outer region; and the O-antigen, a variable polysaccharide chain that imparts serological diversity and modulates surface interactions. This composition renders the outer leaflet highly resistant to hydrophobic compounds while permitting controlled entry.[1] Embedded within the outer membrane are porins, trimeric β-barrel proteins such as OmpF and OmpC in Escherichia coli, which form water-filled channels for passive diffusion of small hydrophilic molecules up to approximately 600 Da, including nutrients like sugars and amino acids. These porins, with 16 β-strands per monomer and exclusion limits tuned by constriction loops, enable essential solute exchange while restricting larger or hydrophobic substances, thus reinforcing the envelope's barrier function.[1]Surface Structures and Motility
Gram-negative bacteria possess diverse surface structures that extend beyond the cell envelope, enabling interactions with environments and facilitating motility. These include flagella for propulsion, pili for adhesion and movement, and polysaccharide-based capsules or slime layers for protection. These appendages are crucial for survival, colonization, and evasion of host defenses.[11] Flagella in Gram-negative bacteria are complex, helical filaments composed of flagellin proteins, anchored via a basal body that spans the inner and outer membranes. They exhibit various arrangements: polar (a single flagellum at one pole, as in Vibrio cholerae), lophotrichous (a tuft of flagella at one or both poles), or peritrichous (multiple flagella distributed around the cell surface). The rotary motor at the base, powered by the proton motive force generated across the inner membrane, drives flagellar rotation at speeds up to 100 Hz, enabling directed movement via chemotaxis toward nutrients or away from toxins. In V. cholerae, the polar flagellum facilitates swimming in aquatic environments, aiding dissemination.[14][15][16][17][18] Pili, also known as fimbriae, are thinner, proteinaceous filaments protruding from the outer membrane. Type 1 pili mediate adhesion to host epithelial cells via tip adhesins like FimH, which bind mannose residues, promoting colonization in pathogens such as uropathogenic Escherichia coli. Type IV pili, retractable and dynamic, drive twitching motility—a jerky, surface-associated movement—by extending, attaching to substrates, and retracting, as seen in Pseudomonas aeruginosa. These pili also facilitate DNA uptake during natural transformation, enhancing genetic diversity.[19][20][21] Capsules and slime layers consist of extracellular polysaccharides tightly associated with the cell surface. Capsules form a discrete, gel-like layer that shields against phagocytosis by host immune cells and desiccation in dry conditions, as in Klebsiella pneumoniae. Slime layers are looser, amorphous coatings providing similar but less rigid protection. These structures contribute to biofilm formation and environmental resilience.[11][22] Motility in Gram-negative bacteria encompasses swimming in liquid media via flagella rotation, producing run-tumble or run-reverse patterns for navigation. Swarming involves coordinated, rapid migration across moist surfaces, often requiring flagella and surfactants, as in Proteus mirabilis. Gliding motility occurs without visible flagella, powered by type IV pili extension-retraction cycles or other mechanisms like the Gld system in Flavobacterium johnsoniae. These modes enable microhabitat exploration and host tissue penetration.[23][18]Classification and Diversity
Taxonomic History and Methods
The classification of Gram-negative bacteria originated in the late 19th century, primarily relying on observable morphological features such as cell shape (e.g., rods, cocci, or spirals) and arrangement, as pioneered by microbiologists like Ferdinand Cohn who established initial genera based on these traits. By the early 20th century, physiological characteristics, including growth requirements, motility, and oxygen tolerance, were incorporated, followed by biochemical tests that assessed enzymatic activities like oxidase and catalase reactions to differentiate subgroups such as enterics from pseudomonads. These phenotypic approaches, while practical for identification in clinical and environmental settings, often grouped bacteria based on superficial similarities rather than evolutionary relatedness.[24][4][25] Bergey's Manual of Determinative Bacteriology, first published in 1923 under the editorship of David Hendricks Bergey, marked a pivotal advancement by compiling a standardized phenotypic framework for bacterial identification, organizing Gram-negative species into informal groups like the Azotobacteraceae and Parvobacteriaceae based on morphology, staining, and biochemical profiles. Through nine editions up to 1994, the manual refined these groupings with increasingly detailed tests, such as sugar fermentation patterns and nitrate reduction, serving as the authoritative reference for determinative taxonomy. The transition to Bergey's Manual of Systematic Bacteriology, beginning in 1984, began integrating emerging phylogenetic insights while retaining phenotypic keys, reflecting the growing recognition of limitations in purely descriptive systems.[26][27] The shift to molecular methods accelerated in the 1970s with Carl Woese's pioneering use of 16S ribosomal RNA (rRNA) sequencing, which provided a stable genetic marker for reconstructing evolutionary histories independent of variable phenotypes. Woese and George Fox analyzed 16S rRNA oligonucleotide catalogs from diverse prokaryotes, including Gram-negative species like Escherichia coli and Pseudomonas aeruginosa, revealing that rRNA sequences evolve slowly enough to resolve deep phylogenetic divergences while capturing fine-scale relationships through similarity metrics. This approach exposed the inadequacies of phenotypic classification, as it prioritized ancestry over convergent traits.[28][29] Phenotypic methods faced significant challenges from convergence, where unrelated Gram-negative bacteria developed similar traits due to environmental pressures or gene transfer, leading to artificial groupings; for instance, oxidase positivity, a key test for aerobes like pseudomonads, appeared in phylogenetically distant taxa, including some anaerobes, obscuring true relationships. Catalase activity similarly converged across lineages, complicating distinctions in early schemes. These issues underscored the need for genotypic tools. A landmark milestone came in 1988 when Stackebrandt et al. formally proposed the phylum Proteobacteria, consolidating disparate Gram-negative groups—such as enterobacteria, rickettsias, and purple nonsulfur bacteria—into a monophyletic clade based on 16S rRNA phylogeny, transforming taxonomic practice.[24]Phylogenetic Classification
Gram-negative bacteria occupy diverse positions within the domain Bacteria, with the phylum Proteobacteria representing the largest and most species-rich group, encompassing the vast majority of described Gram-negative taxa.[30] Other prominent phyla include Bacteroidota, Chlamydiota, Spirochaetota, and numerous additional lineages such as Aquificota and Deferribacterota, reflecting the broad distribution of this phenotype across the bacterial tree of life.[31] The Gram-negative trait is polyphyletic, arising independently in multiple bacterial lineages rather than defining a monophyletic clade, as Gram staining reflects cell envelope architecture rather than shared ancestry.[32] Phylogenetic reconstructions indicate that diderm bacteria—those with a double-membrane envelope typical of Gram-negatives—have evolved convergently from monoderm ancestors on several occasions, challenging earlier models of a single origin for the outer membrane.[32] This polyphyly is further supported by analyses showing independent losses of the outer membrane in monoderm groups like Firmicutes, with Gram-negatives emerging as a convergent adaptation across phyla.[33] Key phylogenetic markers for classifying Gram-negative bacteria include sequences of the 16S rRNA gene, a conserved housekeeping gene present in all bacteria that enables broad-scale evolutionary inference due to its slow mutation rate and universality.[34] Complementing this, whole-genome phylogenomics employs concatenated alignments of ubiquitous single-copy proteins, such as the 120 markers used in the Genome Taxonomy Database, to construct high-resolution trees that mitigate limitations of single-gene approaches like horizontal gene transfer artifacts.[35] These methods delineate major divisions between monoderm (single-membrane, often Gram-positive) and diderm (double-membrane, Gram-negative) bacteria, positioning Gram-negatives firmly within the diderm category based on shared envelope biogenesis genes.[36] Recent post-2020 advancements in metagenomics have profoundly expanded understanding of Gram-negative diversity by recovering genomes from uncultured lineages, revealing novel phyla and deep-branching groups that were previously inaccessible through culture-dependent methods.[37] Genome-resolved metagenomic approaches, including long-read sequencing, have identified thousands of high-quality metagenome-assembled genomes from environmental samples, highlighting uncultured Proteobacteria relatives and other diderm taxa that constitute a substantial portion of global bacterial diversity.[37]Major Phyla and Representative Groups
Gram-negative bacteria exhibit immense phylogenetic diversity, encompassing numerous phyla that inhabit diverse environments from soil and oceans to animal hosts and extreme thermal sites. While the total number of described prokaryotic species is over 25,000 as of 2025, Gram-negative bacteria represent the majority, with the Proteobacteria phylum alone accounting for a substantial portion of known diversity in both environmental and clinical contexts.[38][39] This phylum dominates due to its metabolic versatility, including aerobic, anaerobic, photosynthetic, and nitrogen-fixing capabilities. Other major phyla, such as Bacteroidetes, contribute significantly to anaerobic niches like the gut microbiome, while specialized groups like Chlamydiae and Spirochaetes highlight adaptations to intracellular or motile lifestyles.[40] The Proteobacteria (also known as Pseudomonadota) form the largest and most diverse phylum of Gram-negative bacteria, subdivided into several classes based on 16S rRNA phylogeny. The Alphaproteobacteria class includes oligotrophic species adapted to nutrient-poor environments, with notable representatives like Rhizobium, which forms symbiotic relationships with legumes to fix atmospheric nitrogen via root nodules.[40] Betaproteobacteria are often eutrophic and include human pathogens such as Neisseria, responsible for diseases like gonorrhea (N. gonorrhoeae) and meningitis (N. meningitidis). The Gammaproteobacteria class is the most metabolically and ecologically diverse, encompassing aerobic respirers like Pseudomonas, which thrives in soil and water and causes opportunistic infections (e.g., P. aeruginosa), as well as enteric bacteria such as Salmonella (e.g., S. enterica, associated with foodborne illness). These classes illustrate the phylum's broad ecological roles, from free-living decomposers to symbionts and pathogens.[40] Bacteroidetes (now classified as Bacteroidota) represent another key phylum of Gram-negative bacteria, predominantly anaerobic and specialized in fermenting complex carbohydrates. These rod-shaped organisms are major components of the human gut microbiome, where they aid in polysaccharide breakdown; Bacteroides fragilis, for instance, is a prominent mutualist but can act as an opportunistic pathogen in abscesses. Members of this phylum are bile-resistant and thrive in oxygen-limited habitats like the intestines of animals and sediments.[41] With over 99 described species in the genus Bacteroides alone, the phylum underscores the anaerobic diversity within Gram-negative lineages.[42] Additional phyla highlight further specializations among Gram-negative bacteria. The Chlamydiae (Chlamydiota) phylum consists of obligate intracellular parasites with a unique biphasic life cycle, lacking peptidoglycan but possessing a type III secretion system for host cell invasion; Chlamydia trachomatis is a representative species causing trachoma and sexually transmitted infections.[41] Spirochaetes (Spirochaetota) are helical, motile bacteria propelled by axial filaments, adapted to viscous environments; Treponema pallidum exemplifies this group, causing syphilis through tissue invasion.[41] The Aquificota phylum includes hyperthermophilic, chemolithoautotrophic rods that inhabit extreme hydrothermal vents and hot springs, often oxidizing hydrogen or sulfur; Aquifex aeolicus grows optimally above 80°C and represents early-branching Gram-negative lineages.[43] These phyla collectively demonstrate the adaptive radiation of Gram-negative bacteria across ecological niches.| Phylum | Habitats | Oxygen Requirements | Example Species |
|---|---|---|---|
| Proteobacteria | Soil, water, animal hosts, diverse | Aerobic, facultative, anaerobic | Rhizobium leguminosarum (Alpha), Neisseria gonorrhoeae (Beta), Pseudomonas aeruginosa (Gamma), Salmonella enterica (Gamma) |
| Bacteroidetes | Gut microbiomes, sediments, anaerobic environments | Mostly anaerobic | Bacteroides fragilis |
| Chlamydiae | Intracellular in eukaryotic hosts | Obligate aerobes (host-dependent) | Chlamydia trachomatis |
| Spirochaetes | Aquatic, soil, mucosal surfaces | Mostly microaerophilic or anaerobic | Treponema pallidum |
| Aquificota | Hydrothermal vents, hot springs | Aerobic or microaerophilic (thermophilic) | Aquifex aeolicus |