The Gram-stain technique is used to classify bacteria as either Gram-positive or Gram-negative depending on their colour following a specific staining procedure originally developed by Hans Christian Gram. As the word "Gram" is derived from a name it is always written with an upper case "G".

Following the Gram stain procedure, and on visualization with a microscope Gram-positive bacteria appear dark blue or violet due to the crystal violet stain; Gram-negative bacteria, which cannot retain the crystal violet stain, appear red or pink due to the counterstain. Gram-positive bacteria retain the crystal violet due to a difference in structure of their cell wall, specifically the amount of peptidoglycan.

Gram-negative bacteria do not retain the crystal violet dye in the Gram stain protocol. Gram-negative bacteria will thus appear red or pink following the Gram stain procedure due to the effects of the counterstain (for example safranin).

The cell envelope is defined as the cell membrane and cell wall plus an outer membrane, if one is present. The cell envelope of Gram-negative bacteria contains an outer membrane composed by phospholipids and lipopolysaccharides which face the external environment. The lipopolysaccharides confer an overall negative charge to the Gram-negative cell wall. The chemical structure of the outer membrane lipopolysaccharides is often unique to specific bacterial strains. Many species of Gram-negative bacteria are pathogenic. This pathogenicity is often associated with the lipopolysaccharide layer of the Gram-negative cell envelope.

Gram-negative bacteria have a characteristic cell envelope structure very different from Gram-positive bacteria. Gram-negative bacteria have a cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane containing lipopolysaccharide. There is a space between the cytoplasmic membrane and the outer membrane called the periplasmic space or periplasm. The periplasmic space contains the peptidoglycan.

Genera of Gram-negative bacteria include:

• Acinetobacter
• Actinobacillus
• Bordetella
• Brucella
• Campylobacter
• Cyanobacteria
• Enterobacter
• Erwinia
• Escherichia coli
• Franciscella
• Helicobacter
• Hemophilus
• Klebsiella
• Legionella
• Moraxella
• Neisseria
• Pasteurella
• Proteus
• Pseudomonas
• Salmonella
• Serratia
• Shigella
• Treponema
• Vibrio
• Yersinia

Labels: bacteria, bacteriology, bacterium, gram negative bacteria, gram stain

from Saraswathi Lanka (University of Illinois College of Veterinary Medicine) writing in "Doodys Reviews":
"This broad overview of many aspects of the family Pasteurellaceae provides excellent coverage of the current status of taxonomy and phylogeny of this diverse group of bacteria. ... This is a much needed information resource for researchers. ... This is a rich source of information and provides well balanced coverage of relevant topics. It is a comprehensive guide that provides critical insight into the current understanding of molecular and genomic aspects of Pasteurellaceae" ... read more

Pasteurellaceae: Biology, Genomics and Molecular Aspects
Publisher: Caister Academic Press
Edited by: Peter Kuhnert and Henrik Christensen
Publication date: 2008
ISBN: 978-1-904455-34-9

Labels: bacteria, bacteriology, book review, pasteurella, Pasteurellaceae

Botulinum neurotoxins (BoNTs) are the most potent natural toxins known. The family of BoNTs comprises seven antigenically distinct serotypes (A to G) that are produced by various toxigenic strains of spore-forming anaerobic Clostridium botulinum. They act as metalloproteinases that enter peripheral cholinergic nerve terminals and cleave proteins that are crucial components of the neuroexocytosis apparatus, causing a persistent but reversible inhibition of neurotransmitter release resulting in flaccid muscle paralysis.

Apart from being the sole causative agent of the deadly food poisoning disease, botulism, BoNTs pose a major biological warfare threat due to their extreme toxicity and easy production. Interestingly they also serve as powerful tools to treat an ever expanding list of medical conditions. A better understanding of the structure-function relationship of clostridial neurotoxins will not only help decipher their molecular mode of action but will also provide a greater understanding of the potential use of their individual domains in answering more fundamental questions of neuroexocytosis. It is also critical for designing effective specific inhibitors to counter botulism biothreat, and for the development of new therapeutics.

from Kukreja and Singh in Microbial Toxins: Current Research and Future Trends

Further reading:

1. Microbial Toxins
2. Clostridia: Molecular Biology in the Post-genomic Era

Labels: bacteria, biodefense, botulism, clostridia, clostridium, toxin, toxins

The Type V secretion system was first described twenty years ago. Since then, much work has be done to elucidate functional aspects of members of this family and their mechanisms of biogenesis. What was once considered to be a quirky one-off system, with the discovery of the IgA1 protease secretion system of Neisseria gonorrhoeae, has been revealed as the largest family of secreted proteins amongst the Gram-negative bacteria.

The type V secretion system (T5SS) is comparatively is less complex than types I to IV. This secretion mechanism has been subdivided into sub-types (a), (b) and (c), as variations upon a theme were discovered. Each polypeptide secreted via the T5SS contains an N-terminal Sec-dependant signal sequence required to direct inner membrane export. The significant functional domain classifying molecules into these secretion systems is the dedicated outer membrane (outer membrane) β-barrel protein domain, through which secretion of the effector molecule is achieved.

from Scott-Tucker and Henderson in Bacterial Secreted Proteins

Further reading:

1. Bacterial Secreted Proteins: Secretory Mechanisms and Role in Pathogenesis
2. Microbiology Books

Labels: bacteria, protein, protein secretion, proteins

Type IV secretion systems are multiprotein complexes that mediate the translocation of macromolecules (proteins, DNA or DNA-protein complexes) across the bacterial cell envelope into the extracellular medium or directly into recipient cells. This strategy is exploited for the delivery of effector molecules that modulate host cell interactions by bacterial pathogens and symbionts. Type IV secretion systems also mediate the translocation of DNA molecules from bacteria and the uptake of DNA into bacteria and thereby contribute to horizontal gene transfer.

The term type IV secretion system (T4SS) was originally based on the significant sequence similarities between the protein components of macromolecular transporters used for plasmid transfer and for the delivery of virulence factors from bacterial pathogens to their hosts. The two first examples that prompted the proposal of T4SSs as a distinct family of macromolecular transporters were the conjugative plasmid RP4 transfer machinery (Trb) and the VirB/D4 machinery used by the plant pathogen Agrobacterium tumefaciens for the delivery of single-stranded DNA and of protein virulence factors to plants.

T4SSs carry out one of three functions. The first group of T4SSs translocates exclusively DNA and serves for the uptake or secretion of genetic information. The second group translocates DNA molecules as well as proteins from donor to recipient cells. The third group exclusively translocates proteins and those are either secreted into the exterior medium or directly into recipient cells.

Since the original proposal that T4SS constitute a distinct class of secretion systems was made, the number of known T4SS has continued to grow and both metagenomic as well as functional analyses continue to add new examples. Since many T4SSs localize on plasmids they can be transmitted by horizontal gene transfer and this has very interesting implications for their evolution.

from Christian Baron in Bacterial Secreted Proteins

Further reading:

1. Bacterial Secreted Proteins: Secretory Mechanisms and Role in Pathogenesis
2. Microbiology Books

Labels: bacteria, protein, protein secretion, proteins

The type-III secretion system (T3SS) is an export machine used by pathogenic Gram-negative bacteria to deliver proteins straight into the eukaryotic cytosol with the aim to subvert the host cell defense. After the discovery of T3S in 1990, significant progress has been made in the understanding of its structure, assembly and function. The basic structure consisting of the membrane-embedded basal body, the needle and the tip structure has been analyzed in more detail. The composition of several structural components has been determined and important insights into the assembly process have been gained. The relationship between the T3SS of pathogenic bacteria and the flagellum has been noted. Besides the structural similarities, the assembly of these two nanomachines shows some commonalities, for example the length control of external structures such as the T3 needle and the flagellar hook. The T3SS also includes the pore forming translocator proteins, effector proteins and a set of specific chaperones. A recent review on the type-III secretion system focuses on the structure and assembly of this fascinating nanomachine.

from Sorg and Cornelis in Bacterial Secreted Proteins

Further reading:

1. Bacterial Secreted Proteins: Secretory Mechanisms and Role in Pathogenesis
2. Microbiology Books

Labels: bacteria, protein, protein secretion, proteins

Gram-negative bacteria have evolved several secretory pathways to release proteins or toxic factors into their surrounding environment. Many virulence determinants, including extracellular toxins and proteases, are secreted by the type II secretion system (T2SS) which is widely conserved and common among γ-proteobacteria.

Typical T2SSs are composed of 12 to 16 proteins termed Gsp (General secretion pathway) proteins. These components associate in a multiprotein complex that constitutes a large structure (the secreton) that spans the periplasm and is thought to connect inner and outer membranes. Exoproteins that use the T2SS are characterized by the presence of a leader peptide (or signal peptide) at their N terminus and are secreted in the extracellular medium by a two-step process involving a transient periplasmic intermediate. The T2SS is unique in its ability to promote secretion of large multimeric proteins that are folded in the periplasm. The system is also characterized by a species-specificity, which is mainly related to the GspC and GspD components, the gatekeepers.

Although relatively little attention has been payed to the regulation of T2SSs, it was observed that expression of most of the genes encoding T2SS-dependent exoproteins is growth phase-dependent or strictly regulated by environmental signals. In Pseudomonas aeruginosa, T2SS assembly and most of the T2SS-dependent exoproteins are regulated via quorum sensing, a mechanism that senses the density of a surrounding bacterial population.

Besides typical T2SSs, some secretory systems are found which contain all the T2SS components but in a different genetic organization. Some incomplete systems have also been described which contain genes homologous to T2SS but dispersed on the bacterial chromosome. Components of these systems can either associate with classical T2SS components to constitute a functional hybrid machinery or represent peculiar systems with strictly defined functions.

from Michel and Voulhoux in Bacterial Secreted Proteins

Further reading:

1. Bacterial Secreted Proteins: Secretory Mechanisms and Role in Pathogenesis
2. Pseudomonas: Genomics and Molecular Biology
3. Microbial Toxins: Current Research and Future Trends

Labels: bacteria, protein, protein secretion, proteins, Pseudomonas, toxin

Writting in the latest issue of the ISPP Newsletter published by the International Society for Plant Pathology, Chris Hayward describes the new book on Plant Pathogenic Bacteria from Caister Academic Press as:

"... comprehensive in coverage ... This book is a timely addition to the literature in a rapidly expanding field which provides ample evidence of hypothesis testing on a broad front."

Further reading: Plant Pathogenic Bacteria: Genomics and Molecular Biology

Labels: bacteria, book review, plant

Bacteria have developed numerous systems to secrete proteins or DNA in order to modify their immediate surroundings or to obtain an advantage in a competitive and hostile environment. Since Gram-negative bacteria possess two membranes, the inner (cytoplasmic) membrane and the outer membrane, transport machines for protein secretion have the challenging task of circumventing two barriers to reach the exterior. A rather simple transport apparatus, the Type I secretion machinery, composed of only three proteins residing in the inner and outer membrane of Gram-negative bacteria achieve this objective in a single step. The Type I secretion pathway although also present in Gram-positive bacteria, has been analysed in greatest detail in Gram-negative bacteria. Almost all Type I transport substrates are polypeptides, varying from the small Escherichia coli peptide colicin V, (10 kDa) to the large Pseudomonas fluorescens cell adhesion protein LapA of 900 kDa. While these two examples reflect the range of the size of Type I transport substrates, the best characterized are the RTX toxins and the lipases. Type I secretion is also involved in export of non-proteinaceous substrates like cyclic β-glucans or polysaccharides.

from Jenewein et al in Bacterial Secreted Proteins

Further reading:

1. Bacterial Secreted Proteins: Secretory Mechanisms and Role in Pathogenesis
2. Pseudomonas: Genomics and Molecular Biology
3. Microbial Toxins: Current Research and Future Trends

Labels: bacteria, protein, protein secretion, proteins, Pseudomonas, toxin

A description of the twin-arginine translocation (Tat) pathway continues our series on protein secretion in microorganisms.

The twin-arginine translocation (Tat) pathway is a protein transport system in bacteria, archaea and chloroplasts with the ability to export proteins in a fully folded conformation. Proteins are targeted to the Tat pathway by an N-terminal signal peptide containing an almost invariant twin-arginine sequence motif. Pretranslocational folding is necessitated by the incorporation of metallo-cofactors, assembly into oligomeric complexes, and presumably rapid folding kinetics. Many Tat systems comprise three functionally individual membrane proteins, termed TatA, TatB, and TatC, whereas especially Gram-positive bacteria possess minimal TatAC translocases, in which TatA functionally replaces TatB. TatC and TatB form a complex that is involved in recognition of Tat signal sequences and their insertion into the membrane. TatA mediates the actual translocation event, but it is unclear whether it does so by forming the pore-like structures that it displays when purified to homogeneity. Energy is derived from either component of the proton-motive force, ΔpH or ΔΨ, and is required only for late steps following signal sequence cleavage. Substrates that either lack the twin-arginine pair or are in a malfolded conformation in general are not translocated. The mechanisms by which non-functional substrates are rejected are not understood. For cofactor-containing substrates, proof-reading seems to depend on the activity of specific cytosolic chaperones.

from Panahandeh et al in Bacterial Secreted Proteins

Further reading: Bacterial Secreted Proteins: Secretory Mechanisms and Role in Pathogenesis

Labels: archaea, bacteria, protein, protein secretion, proteins