LIPID-CELL WALL IDENTIFICATION

Carl G. Buscato and Riza MaeEayteBio 135

Republic of the PhilippinesCollege of Natural Science and

MathematicsMINDANAO STATE UNIVERSITY

General Santos City

Classifications Schemes to Consider

Gram Reaction

Gram Positive Gram Negative

Acid Fast

Non-acid Fast

Gram Positive Acid Fast

Gram Positive Non-Acid Fast

Mycolic acid (60%)

Have lipids linked to peptidoglycan

Includes the genera of: Mycobacteria (C60-90) Nocardia (C46-58) Rhodococcus (C28-48) Corynebacterium (C24-36)

Gram Positive Acid Fast

Thick, multi-layered peptidoglycan

Absence of lipid outer membrane

Low lipid, lipoprotein and lipopolysaccahride content

Produces exotoxins

Gram Positive Non-Acid Fast

Gram Negative

Thin, single-layered peptidoglycan layer

Presence of lipid outer membrane

High lipid, lipoprotein and lipopolysaccharide content

Produces endotoxin (Lipid A) and exotoxins

Since, gram negative have high lipid content, the lipid-cell wall taxonomic scheme best fits for them

Gram Negative

Lipids Water-soluble (polar) organic molecules

that are important for the structure of the cytoplasmic membrane and the cell wall. Lipids are a useful non-genetic criteria to differentiate Archaea from Bacteria.

Classified into: Phospholipid Phosphatyl-glycerol Di-phosphatyl-glycerol Phosphatyl-ethanolamine Phosphatyl-choline

Lipid Classification Phospholipid

Lipids containing a substituted P-group and 2 fatty acid chains on a glycerol backbone. Archaea and Bacteria are distinguished by the bond between the FA tail and the glycerol head; ether-bond (Archae), esther-bond (Bacteria)

Lipid Classification Phosphatyl-glycerol

Phospholipid with an attached gylcerol molecule CHOH(CH2OH)2 to the P-group

Distinguishes microbes according to their configuration: diacyl (I 60% in E. coli), alklacyl ( 20% in Salmonella typhirum) and alkenacyl (30% in Corynebacterium amycolatum)

Phosphatyl-choline Phospholipids with an attached choline

molecule OH(CH2)2N(CH3)3 to P-group Phosphatyl-ethanolamin

Phospholipids with an extra ethanolamine molecule OH(CH2)2NH3+ to the P-group

Di-phosphatyl-glycerol (cardiolipin) Two phospholipids bound together by a

glycerol molecule CHOH(CH2OH)2

Lipid Classification

Lipid Classification

Peptidoglycan Classification

Fatty Acid Classification Branched

anteiso, asymmetrically at different sites branched

iso, symmetrically branched methyl, H swapped with CH3 group.

Cyclopropane FA-chain is interrupted by a ring of C-atoms

(C3H8). Hydroxy

H swapped w/ OH group of some FA-C-atoms; e.g.: 2-OH,: 3-OH; in Archaea periodically branched CH3 side chains.

Saturated No double bond b/w any C of the FA-chain;

all C-atoms are saturated w/ H-atoms Poly-Unsaturated FA

Have one or more positions along the FA-polymer chain where 2 adjacent C are linked by double bond (2 shared pairs of electrons); consequently, fewer Hs are bonded to the Cs.

Fatty Acid Classification

Chemotaxonomy Phenotypic analysis based on

classification of bacterial cell wall constitute (chemical markers such as lipids, proteins and sugars)

Classification and identification can be done through differences on distribution, configurations and absence/presence of molecules per species/genera

Chemotaxonomic methods include:

Chemotaxonomic Methods: Thin Layer Chromatography The classic method of

polar lipid extraction utilizes a monophasic mixture of chloroform, methanol and water for extraction.

2D thin layer chromatography can be used to determine simple 2-dimensional patterns of polar lipids which may be characteristic of individual taxa.

Chemotaxonomic Methods: Fatty Accid Methyl Ester (FAME) Analysis

Chemotaxonomic Methods: MALDI-TOF MS

Chemotaxonomic Methods: MALDI-TOF MS

Chemotaxonomic Methods: MALDI-TOF MS

MALDI-TOF MS: (Mass Spectronomy)

MALDI-TOF MS: (MALDI)

MALDI-TOF MS: (TOF)

Thought to ponder: