Gram negative bacteria

Gram-negative bacteria constitute a diverse and ecologically widespread group of prokaryotes defined by their distinctive cell envelope, which includes a thin peptidoglycan layer situated between an inner cytoplasmic membrane and an outer membrane containing lipopolysaccharide. This structural arrangement causes Gram-negative organisms to lose the crystal violet stain during the Gram staining procedure and take up a counterstain, appearing pink or red under the microscope. These bacteria occupy almost every environment capable of supporting life, and the group includes both beneficial environmental species and significant human, animal, and plant pathogens.

Structural Characteristics

The defining feature of Gram-negative bacteria is their diderm (two-membrane) cell envelope. Key structural components include:

• Inner (cytoplasmic) membrane: a phospholipid bilayer that encloses the cytoplasm.
• Thin peptidoglycan layer: located in the periplasmic space; considerably thinner than in Gram-positive bacteria.
• Outer membrane: a lipid bilayer asymmetrically composed of phospholipids on the inner leaflet and lipopolysaccharides (LPS) on the outer leaflet.
• Lipopolysaccharide: consisting of lipid A, a core polysaccharide, and the O-antigen. Lipid A functions as an endotoxin and can trigger strong immune responses, including septic shock during bacterial lysis.
• Periplasm: the gel-like space between the membranes containing enzymes and transport proteins.
• Porins: protein channels in the outer membrane that regulate the passage of nutrients and small molecules.
• Braun’s lipoprotein: a structural component linking the outer membrane to the peptidoglycan, providing additional stability.

Gram-negative bacteria lack teichoic and lipoteichoic acids. Their flagellar arrangement typically involves four supporting rings, in contrast to the two-ring system of Gram-positive bacteria. Most Gram-negative species do not form endospores.
Although the conventional Gram-negative organisms are unified by the presence of an LPS-containing outer membrane, some bacteria outside this core group stain Gram-negative but possess distinctive structures, such as the mycolic acid-rich outer layers of Mycobacteriales.

Antibiotic Resistance and Medical Challenges

The outer membrane of Gram-negative bacteria presents a significant barrier to many antibiotics, detergents, and host defence enzymes such as lysozyme. As a result, Gram-negative pathogens are prominent contributors to antimicrobial resistance. The endotoxin activity of lipid A presents additional clinical risks, as its release during bacterial destruction can initiate life-threatening conditions, including septic shock with hypotension, respiratory failure, and metabolic acidosis.
Several classes of antibiotics target Gram-negative organisms. Broad-spectrum agents include aminopenicillins, ureidopenicillins, cephalosporins, carbapenems, quinolones, antifolates, and β-lactam/β-lactamase inhibitor combinations. More selective activity is provided by monobactams such as aztreonam and by aminoglycosides and fluoroquinolones like ciprofloxacin. Many of these drugs also retain activity against Gram-positive organisms, although the reverse spectrum is less common.

Taxonomy and Evolutionary Background

Historically, bacteria were classified into Gram-positive and Gram-negative groups based on staining results, but modern molecular phylogenetics has revealed the limitations of this approach. Gram staining does not reliably indicate evolutionary relationships, as structurally similar cell envelopes have evolved independently in some lineages.
Two major categories of diderm bacteria are recognised:

• Conventional LPS-diderm bacteria: These groups share a common ancestor and are unified by conserved molecular signatures, including characteristic insertions in the HSP60 (GroEL) protein. They are placed within the kingdom Pseudomonadati (formerly Hydrobacteria) and include phyla such as Pseudomonadota, Chlamydiota, Bacteroidota, Cyanobacteria, and Spirochaetota.
• Atypical diderm bacteria: These include groups such as the Negativicutes and Fusobacteriota, which show diderm structures yet cluster phylogenetically within traditionally monoderm (Gram-positive) phyla. They lack the molecular signatures that characterise the LPS-diderm clade. Mycobacteriales also possess an outer membrane, but it is composed of mycolic acids rather than LPS, giving rise to specific staining behaviours.

The ancestral lineage of bacteria is proposed to have possessed a single membrane (monoderm structure), with the outer membrane of Gram-negative bacteria evolving later—possibly as an adaptive response to environmental pressures, including exposure to naturally occurring antibiotics.
Some species such as Deinococcus show intermediate characteristics, staining Gram-positive due to their thick peptidoglycan layer while also possessing an outer membrane, making them important in evolutionary studies of bacterial envelope development.

Ecological Diversity and Notable Groups

Gram-negative bacteria are abundant in soil, freshwater, marine environments, and within host organisms. They play essential roles in nutrient cycling, photosynthesis, and symbiosis.
Major Gram-negative taxa include:

• Proteobacteria: a large superphylum that includes Escherichia coli, Salmonella, Pseudomonas, Moraxella, Legionella, Bdellovibrio, and numerous other genera.
• Cyanobacteria: photosynthetic organisms that contribute to oxygen production and form the basis of many aquatic food webs.
• Spirochaetes: including pathogenic species such as Treponema pallidum and Borrelia burgdorferi.
• Green sulfur bacteria and other specialised phototrophs.

Clinical Importance and Pathogenic Species

Many medically important pathogens are Gram-negative, including:

• Diplococci:
  • Neisseria gonorrhoeae (sexually transmitted infection)
  • Neisseria meningitidis (meningococcal meningitis)
  • Moraxella catarrhalis (respiratory infections)
• Coccobacilli:
  • Haemophilus influenzae (respiratory tract infections)
• Bacilli (rod-shaped species):
  • Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens
  • Pseudomonas aeruginosa, Legionella pneumophila (respiratory diseases)
  • Helicobacter pylori, Salmonella enteritidis, Salmonella typhi (gastrointestinal infections)

Certain Gram-negative bacteria are frequently implicated in hospital-acquired infections, including Acinetobacter baumannii, a major cause of bacteraemia, ventilator-associated pneumonia, and secondary meningitis in intensive care settings.

Biological Processes: Transformation and Genetic Exchange

Gram-negative bacteria participate in horizontal gene transfer mechanisms such as transformation, conjugation, and transduction. These processes significantly enhance adaptability and play a major role in the spread of antibiotic resistance genes among pathogenic species.