Altered Growth and Envelope Properties of Polylysogens Containing Bacteriophage Lambda N−cI− Prophages
Abstract
:1. Introduction
2. Results
2.1. Envelope-associated Properties of the Polylysogen
2.1.1. Properties Associated with the Cytoplasmic Membrane
2.1.2. Properties Associated with the Peptidoglycan and the Outer Membrane
2.1.3. Envelope Composition
2.2. Characteristics of the Polylysogen Filaments
2.2.1. Filament Length
2.2.2. Irregular Placement and Uniqueness of Septa in the Polylysogen Filament
2.2.3. Division Status in Different Segments of the Polylysogen Filament
2.3. Deconversion of the Polylysogen
2.4. Role of Lambda Genes in Conversion
3. Summary and Discussion
4. Materials and Methods
4.1. Chemicals, Bacteria, and Phages
4.2. Methods
4.2.1. General and Common Procedures
4.2.2. Visualization and Microscopy of Bacteria
4.2.3. Isolation and Analysis of Bacterial Envelope and Its Constituents
4.2.4. Quantification of Prophage Copies in Lysogens
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ATP | Adenosine triphosphate |
cAMP | Cyclic AMP |
CFU | Colony-forming unit |
DMSO | Dimethyl sulfoxide |
DNA | Deoxyribonucleic acid |
DOC | Deoxycholate |
E. coli | Escherichia coli |
EDTA | Ethylenediaminetetraacetic acid |
E.O.P. | Efficiency of plating |
FAD | Flavin adenine dinucleotide |
Glc | Glucose |
GlcN | N-acetyl glucosamine |
Hep | Heptose |
HPr | Histidine-containing protein (Phosphocarrier protein) |
KDO | 2-Keto-3-deoxy-octonate |
LDH | Lactate dehydrogenase |
LPS | Lipopolysaccharide |
MTT | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) |
NADH | Nicotinamide adenine dinucleotide |
OL | Operator, left |
OR | Operator, right |
PAGE | Polyacrylamide gel electrophoresis |
PEP | Phosphoenolpyruvate |
PMS | Phenazine methosulfate |
PL | Promoter, left |
pNPP | Para-nitrophenylphosphate |
PR | Promoter, right |
PTS | Phosphotransferase enzyme system |
Rha | Rhamnose |
RNA | Ribonucleic acid |
SDS | Sodium dodecyl sulfate |
TCA | Trichloroacetic acid |
TEMED | Tetramethylethylenediamine |
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Bacteria | Activity / Property Assayed 1 | |||
---|---|---|---|---|
PTS activity a Total (in frozen-thawed cells) | Amount exposed in saline-washed cells | % of total exposed in saline-washed cells | ||
Nonlysogen | 43.3 ± 3 | 0.53 ± 0.02 | 1.20 | |
Polylysogen | 16.6 ± 2 | 9.70 ± 0.8 | 60.5 | |
Uptake of: | ||||
14C-Glucose | 14C-Galactose | 3H-Uridine | ||
Nonlysogen | 3.60 × 104 | 3.6 × 103 | 1.93 × 104 | |
Polylysogen | 2.15 × 104 (60%) | 2.3 × 103 (63%) | 1.00 × 104 (52%) | |
Enzyme activity in membrane fraction | ||||
NADH oxidase b | FAD-linked LDH b | Dye reductase b | ATPase c | |
Nonlysogen | 26.5 ± 2 | 8.00 ± 0.2 | 14.5 ± 1 | 28.0 ± 1 |
Polylysogen | 19.0 (60.1%) | 4.08 (57%) | 8.5 (58%) | 14.00 (50%) |
Enzyme | % Total Activity Released by EDTA Treatment 1 | |
---|---|---|
Nonlysogen | Polylysogen | |
(a) Periplasmic: | ||
Alkaline phosphatase | 1.8 | 32.0 |
5′-nucleotidase | 8.0 | 47.0 |
Acid phosphatase | ND | 30.0 |
(b) Cytoplasmic: | ||
G6PD | ND | ND |
Inorganic pyrophosphatase | 9.0 | 10.0 |
Phage | Efficiency of Plating, Relative to E. coli 594 2 | |
---|---|---|
Monolysogen 594 (λ+) | Polylysogen 594 (λN-cI-) | |
T4D | 0.8 | 0.2 |
T3 | 0.9 | 1.0 |
T7 | 1.0 | 1.1 |
Bacteria | Total LPS (% of Dry Envelope, w/v) | Carbohydrate (% of dry LPS, w/w) | |||||
---|---|---|---|---|---|---|---|
Monosaccharides | |||||||
Total | Rha | GlcN | Glc | Hep | KDO | ||
Nonlysogen | 5.0 (100) | 31.3 | 0.92 | 6.5 | 11.1 | 18.6 | 4.22 |
Polylysogen | 3.6 (72) | 32.8 | 0.97 | 6.0 | 11.9 | 18.45 | 4.58 |
Major Peak# | ~Mr/1000 | Area of the Peak * | Ratio (b/a) | |
---|---|---|---|---|
Nonlysogen (a) | Polylysogen (b) | |||
7 | 57.0 | 210 | 235 | 1.10 |
9 | 39.0 | 141 | 82 | 0.58 |
11 | 30.0 | 214 | 360 | 1.60 |
14 | 18.0 | 60 | 123 | 2.05 |
15 | 14.5 | 435 | 99 | 0.22 |
17 | 8.0 | 93 | 108 | 1.16 |
Bacteria | Colony Size | Cell Length | % AP Leakage | E.O.P. of T4 | λDNA Copy |
---|---|---|---|---|---|
Nonlysogen a | Large | Small rod (1×) | 2.4 | 1.0 | 0 |
Deconverted | Large | Small rod (1×) | 3.2 | 0.9 | 0 |
Semi-deconverted | Medium | Short filament (3–4×) | 12.0 | 0.48 | 16 ± 1 |
Polylysogen | Small | Long filament (14–25×) | 32.0 | 0.2 | 22 ± 2 |
Lysogen | Colony Size | Cell Morphology | PTS Activity (% of 594) | % AP Leakage | E.O.P. of T4 (Fraction of 594) | λDNA Copy |
---|---|---|---|---|---|---|
594(λN-cI-) | Small a | Filament, 15× | 39.0 | 32 ± 4 | 0.20 | 24 ± 4 |
594(λN-cI-R-) | Small | Filament, 15× | 34.6 | 30 ± 3 | 0.12 | 22 ± 3 |
594(λN-cI-S-) | Small | Filament, 15× | 34.4 | 29 ± 5 | 0.10 | 24 ± 4 |
594(λN-cI-R-S-) | Small | Filament, 15× | 24.1 | 32 ± 2 | 0.13 | 23 ± 4 |
594(λN-cI-O-) | Large | Short filament, 2× | ND | 4.9 ± 1 | 0.65 | 8 ± 1 |
594(λN-cI-P-) | Large | Short filament, 2× | ND | 7.8 ± 0.4 | 0.60 | 8 ± 1 |
594 | Large | Small rod, 1× | 100 | 1.8 ± 0.1 | 1.00 | 0 |
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Barik, S.; Mandal, N.C. Altered Growth and Envelope Properties of Polylysogens Containing Bacteriophage Lambda N−cI− Prophages. Int. J. Mol. Sci. 2020, 21, 1667. https://doi.org/10.3390/ijms21051667
Barik S, Mandal NC. Altered Growth and Envelope Properties of Polylysogens Containing Bacteriophage Lambda N−cI− Prophages. International Journal of Molecular Sciences. 2020; 21(5):1667. https://doi.org/10.3390/ijms21051667
Chicago/Turabian StyleBarik, Sailen, and Nitai C. Mandal. 2020. "Altered Growth and Envelope Properties of Polylysogens Containing Bacteriophage Lambda N−cI− Prophages" International Journal of Molecular Sciences 21, no. 5: 1667. https://doi.org/10.3390/ijms21051667