Bacteriophages of LAB control measures and significance
Bacteriophages of LAB: Control measures and their significance Prasanta Kumar Choudhury National Dairy Research Institute Karnal, Haryana, 132001
Bacteriophages and Dairy industry• The occurrence of bacteriophage (phage) in dairy starter cultures was first reported in the 1930s by Whitehead and Cox (1935) in Lactococcus starter used for cheddar cheese.• The existence of phage is a recognized problem in the dairy industry.• But the LAB have some phase resistant mechanisms
Phage resistance mechanisms of LABPhage resistant mechanisms of LAB:1. Adsorption Inhibition2. DNA injection blocking3. Restriction modification4. Abortive inhibition
1. Adsorption inhibitionAttachment of a phage to the cell surface is a veryspecific process, dependent on : • Phage specificity • Accessibility of bacterial receptor • Physiochemical properties of the cell envelope • The electrical potential across the cytoplasmic membrane
• The surface of Lc. lactis was covered predominantly by polysaccharides and proteins in the ratio 2 : 1• The composition/ structure of the polysaccharides most likely varies between different strains.• The phages first recognize a polysaccharide before they attach to a protein located in the plasma membrane• The polysaccharides are often composed of rhamnose, glucose, galactose, and glucosamine and are covalently linked to the peptidoglycans
• An S-layer composed of a protein covered the surface of L. helveticus strains.• Change in the amino acid sequence can affect phage adsorption• Failure of a phage to adsorb to a host cell may be due to either the lack of appropriate polysaccharides on the cell surface or to a physical masking of the receptor polysaccharide or protein
• Polysaccharides may either be excreted into the environment as exopolysaccharides (EPS) or form capsular polysaccharides (CPS)• It has been discovered that production of EPS in some cases blocks adsorption, but not always CPS provide bacteria with a higher degree of protection than EPS
2. DNA injection blocking• DNA injection inhibition occurs when phage adsorbs to the cell surface but phage DNA stays inside the head section and fails to enter the host cell cytoplasm.• DNA injection inhibition resulted from an alteration in plasma membrane components of the host cell.• This resistance mechanism appears to be rare (Dinsmore and Klaenhammer, 1995).
3. Restriction/Modification• An R/M system first recognizes a specific DNA sequence; then it either modifies DNA by a methyltransferase or cleaves DNA with a restriction endonuclease• Methylation is performed either on adenine or cytosine located within the recognition sequence by transfer of a methyl group from S-adenosyl-L- methionine• The bacterium methylates its own DNA, thereby protecting it against restriction
• Cleavage by the restriction endonuclease will take place either within or nearby the recognition site or randomly Therefore the R/M system can protect bacteria against foreign DNA such as phages by cleaving invading phage DNA• It only requires that the recognition sequence specific for the R/M system is present in the phage DNA and that the sequence has not been modified by methylation• The DNA of progeny phages that has escaped restriction by an R/M system will be methylated, and therefore these phages can circumvent that specific R/M system and reinfect the bacteria with a high efficiency
4. Abortive inhibition• Abortive infection is a type of phage resistance resulting in decreased production of virulent phages by infected cells but not involving restriction or modification.• Abortive infection results in cell death, but because phage replication is much reduced, the phage population does not increase sufficiently to affect culture activity.• Abortive infection does not induce genetic changes in the infecting phage.
• Most of them are plasmid-encoded. All Abi systems are characterized by an unusually low G + C content of their genes (26–29%)• The AbiA, AbiK, AbiF, and AbiR systems act on the phage DNA replication• AbiG, AbiU, and an undefined Abi system encoded by plasmid pBU1-8 from Lc. lactis subsp. lactis Bu2 inhibits or delays transcription of phage DNA• AbiB was shown to prevent phage growth by promoting degradation of transcripts derived from phage bIL170 (936 species) infection of Lc. lactis subsp. lactis IL1403 starting 10– 15 minutes after infection
• In cells harboring AbiQ, the immature concatemeric form of phage DNA accumulated in the cells. This suggests that it may be defective and unable to be processed into mature phages or that genes involved in phage morphogenesis are affected by AbiQ• It has been suggested that one of the late mRNAs or proteins activates the AbiT mechanism and causes premature cell death
Abortive Infection Mechanisms Characterized from Lactococcus
Problems caused by bacteriophages• Slowing of starter or starter failure• Poor acid development• Decrease in flavor production• Poor rennet action• Poor texture formation Great loss to dairy industry
PHAGE CONTROL Artificial Phage-Resistance Methods1. Use of Phage-Inhibitory Media2. Culture Rotation3. Development of phage resistant mutants4. Encapsulation of starter cultures5. Dairy plant and cleanliness
1. Phage Inhibitory Media• Growth of phages during production of bulk starter can be controlled by using phage-inhibitory media.• These media rely on the ability of phosphate and citrate salts to bind ionic calcium, thus inhibiting phagic absorption (Reiter, 1956).• Phage-control media often contain deionized whey, protein hydrolysates, ammonium and sodium phosphate, citrate salts, and other growth stimulants such as yeast extract (Whitehead, 1993) which prevents phage proliferation.
2. Culture Rotation• Culture rotations control bacteriophagic infection by limiting the length of time that a specific strain or mixture of strains is used.• Cultures following each other in the series are susceptible to different phage types and are therefore unaffected by phages that may have infected the previous culture.• Cultures can be rotated on a daily basis or after each vat of milk is inoculated.
• However, the use of a limited number of cultures at any one time is recommended to reduce exposure to prophages and maintain product uniformity.• Culture rotation does not eliminate phage growth in cheese milk in vats, but if phage numbers are kept to less than 10,000 pfu/mL of cheese whey, acid production is not affected (Huggins, 1984).• Success of a culture rotation is limited by availability of phage-unrelated strains with acceptable fermentation properties. In addition, using many different cultures can result in lack of product uniformity.
3. Development of Phage resistant mutants I. Antisense RNA Strategies II. Utilization of Origin of Replication (PER) III. Utilization of the Phage Repressor
I. Antisense RNA Strategies• A gene is cloned behind a promoter in its antisense orientation• It is anticipated that its antisense RNA transcript will bind to target sense mRNA, preventing translation either by destabilizing and making it more susceptible for degradation by RNases or by inhibiting loading of ribosomes
II. Utilization of Origin of Replication (PER)• Another approach is based on utilization of the phage origin of replication• The method was designated “PER” for phage-encoded resistance• When the cloned origin of replication (ori) of the lactococcal phage Ф50 was introduced into Lc. lactis subsp. lactis NCK203, the host’s insensitivity to Ф50 infection was enhanced as indicated by a reduction in EOP and in plaque size• It was assumed that the cloned ‘per’ locus competes with normal phage replication.
III. Utilization of the Phage Repressor• Temperate phage have a gene encoding a repressor, CI, suppressing the expression of the lytic cycle• Integration of the cI gene of phage A2 into the Lb. casei host chromosome resulted in stable resistance against super infection with the A2 phage
CRISPR DNA• CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are loci containing multiple short direct repeats that are found in the genomes of approximately 40% of bacteria and 90% of archaea.• Prokaryotic immune system confering resistance to exogenous genetic elements such as plasmids and phage.• Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a memory of past exposures.• CRISPR spacers are then used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms
Clustered Regularly Interspaced Short Palindromic Repeats
4. Encapsulation of Dairy Starters• Encapsulation of the dairy starters with effective base materials can prevent the cultures from phage attack.• Calcium alginate are commonly used to preparing encapsulated beads for starter preservation
4. Dairy plant and Cleanliness• Phage development in these growth niches is controlled by effective sanitation.• Phages are disseminated throughout the dairy plant by aerosol and human carriers.• When preparing bulk starter, air drawn into the tank when the culture medium cools should be filter sterilized. Milk in cheese vats is most susceptible to phage contamination during ripening and setting, so these processes should be accomplished in closed systems.
• Air entering cheese manufacturing rooms should be under positive pressure of high-efficiency particulate air (HEPA) filtered air.• Whey should be removed to a physically separate facility, because whey processing produces aerosols that can carry phage particles.• Plant personnel with exposure to whey should not be allowed access to the milk-ripening or bulk starter facilities.