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6.2D: Bacterial Differentiation - Biology

6.2D: Bacterial Differentiation - Biology


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Several bacteria alter their morphology in response to the types and concentrations of external compounds.

Learning Objectives

  • Explain bacterial differentiation to eukaryotic-like sturctures

Key Points

  • Bacterial morphology changes help to optimize interactions with cells and the surfaces to which they attach.
  • Oxidative stress, nutrient limitation, DNA damage and antibiotics exposure are some of stress conditions to which bacteria respond, altering their DNA replication and cell division.
  • The most frequent shape alteration may be filamentation triggered by a limitation in the availability of one or more nutrients.

Key Terms

  • cytoskeleton: A cellular structure like a skeleton, contained within the cytoplasm.
  • septum: a partition that separates the cells of a (septated) fungus
  • segrosomes: multiprotein complexes that partition chromosomes/plasmids in bacteria.
  • cell division: a process by which a cell divides into two cells.

Bacterial morphological plasticity refers to evolutionary changes in the shape and size of bacterial cells. As bacteria evolve, morphological changes occur to maintain the consistency of the cell. However, this consistency could be affected in some circumstances (such as environmental stress) and changes in bacterial shape and size. In bacteria, the transformation into filamentous organisms have been recently demonstrated. These are survival strategies that affect the normal physiology of the bacteria in response to factors such as innate immune response, predator sensing, quorum sensing and antimicrobial signs.

Normally, bacteria have different shapes and sizes which include coccus, rod and helical/spiral (among others less common). This forms the basis for their classification. For instance, rod shapes may allow bacteria to attach more readily in environments with shear stress (e.g., in flowing water). Cocci may have access to small pores, creating more attachment sites per cell and hiding themselves from external shear forces. Spiral bacteria combine some of the characteristics of cocci (small footprints) and of filaments (more surface area on which shear forces can act) and the ability to form an unbroken set of cells to build biofilms. Several bacteria alter their morphology in response to the types and concentrations of external compounds. Bacterial morphology changes help to optimize interactions with cells and the surfaces to which they attach. This mechanism has been described in bacteria such as Escherichia coli and Helicobacter pylori.

Oxidative stress, nutrient limitation, DNA damage and antibiotics exposure are some stress conditions to which bacteria respond, altering their DNA replication and cell division. Filamentous bacteria have been considered to be over-stressed, sick and dying members of the population. However, the filamentous members of some communities have vital roles in the population’s continued existence, since the filamentous phenotype can confer protection against lethal environments.Filamentous E. coli can be up to 70 µm in length and has been identified as playing an important role in pathogenesis in human cystitis. There are different mechanisms identified in some bacteria that are attributable to the development of filamentous forms.

Nutritional stress can change bacterial morphology. The most frequent shape alteration may be filamentation triggered by a limitation in the availability of one or more nutrients. Since the filament can increase a cell’s uptake–proficiency surface without changing its surface-to-volume ratio appreciably, this may be enough reason for cells to be filament. Moreover, the filamentation benefits bacterial cells attaching to a surface because it increases specific surface area in direct contact with the solid medium. In addition, the filamentation may allow bacterial cells to access nutrients by enhancing the possibility that the filament will be exposed to a nutrient-rich zone and pass compounds to the rest of the cell’s biomass. For example, Actinomyces israelii grows as filamentous rods or branched in the absence of phosphate, cysteine, or glutathione. However, it returns to a regular rod-like morphology when adding back these nutrients.


Bacteria vs. Virus

Bacteria are single-celled, prokaryotic microorganisms that exist in abundance in both living hosts and in all areas of the planet (e.g., soil, water). By their nature, they can be either "good" (beneficial) or "bad" (harmful) for the health of plants, humans, and other animals that come into contact with them. A virus is acellular (has no cell structure) and requires a living host to survive it causes illness in its host, which causes an immune response. Bacteria are alive, while scientists are not yet sure if viruses are living or nonliving in general, they are considered to be nonliving.

Infections caused by harmful bacteria can almost always be cured with antibiotics. While some viruses can be vaccinated against, most, such as HIV and the viruses which cause the common cold, are incurable, even if their symptoms can be treated, meaning the living host must have a strong enough immune system to survive the infection.


Bacteria are the oldest organisms known to exist on earth. They belong to the oldest Kingdom Monera while Protists are classified as Kingdom Protista. They have organisms which show characteristics similar to animals, plants or fungi. Thus, they further divided into 3 categories i.e. plant-like Protists, animal-like Protists or fungi-like Protists.

Bacteria are single-celled organisms and their cell structure is very simple. There is no nucleus which is the chief controller of a cell. The DNA, which is the genetic material, is scattered in the cell. Since they do not contain a nucleus, they are known as prokaryotic organisms. They do not contain any small specialized smaller organs known as organelles. They can be rod shaped, spiral, spherical, chain like, etc.

Protists can be either single-celled or multicellular. They contain a nucleus as well as specialized smaller organelles. In addition, their genetic material is compact within an envelope.


6.2D: Bacterial Differentiation - Biology

a School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK

b School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK

c Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, UK
E-mail: [email protected]
Tel: +44 (0)151 795 7689

d Department of Earth and Environmental Sciences, University of Manchester, Manchester, UK

e EM Core Facility, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK

Abstract

The Gram-negative bacterial pathogen Campylobacter jejuni is a major cause of foodborne gastroenteritis worldwide. Rapid detection and identification of C. jejuni informs timely prescription of appropriate therapeutics and epidemiological investigations. Here, for the first time, we report the applicability of Raman spectroscopy, surface-enhanced Raman scattering (SERS) and matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF-MS) combined with chemometrics, for rapid differentiation and characterisation of mutants of a single isogenic C. jejuni strain that disrupt the production of prominent surface features (capsule, flagella and glycoproteins) of the bacterium. Multivariate analysis of the spectral data obtained from these different physicochemical tools revealed distinctive biochemical differences which consistently discriminated between these mutants. In order to generate biochemical and phenotypic information from different locations in the cell–cell wall versus cytoplasm – we developed two different in situ methods for silver nanoparticle (AgNP) production, and compared this with simple mixing of bacteria with pre-synthesised AgNPs. This SERS trilogy (simple mixing with premade AgNPs and two in situ AgNP production methods) presents an integrated platform with potential for rapid, accurate and confirmatory detection of pathogenic bacteria based on cell envelope or intracellular molecular dynamics. Our spectral findings demonstrate that Raman, SERS and MALDI-TOF-MS are powerful metabolic fingerprinting techniques capable of discriminating clinically relevant cell wall mutants of a single isogenic bacterial strain.


Acknowledgements

This research was largely supported by the Lawrence Berkeley National Laboratory (LBNL) Genomes to Watershed Scientific Focus Area funded by the US Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research (OBER) under contract no. DE-AC02-05CH11231. Additional support was provided by LBNL EFRC award no. DE-AC02-05CH11231, NASA NESSF grant no. 12-PLANET12R-0025 and NSF DEB grant no. 1406956, DOE OBER grant no. DOE-SC10010566, Office of Naval Research grants nos. N00014-07-1-0287, N00014-10-1-0233 and N00014-11-1-0918, and by the Thomas C. and Joan M. Merigan Endowment at Stanford University. In addition, funding was provided by the Ministry of Economy, Trade and Industry of Japan. The authors thank J. Eisen for comments, S. Venn-Watson, K. Carlin and E. Jensen (US Navy Marine Mammal Program) for dolphin samples, K.W. Seitz for sequence submission assistance, and the DOE Joint Genome Institute for generating the metagenome sequence via the Community Science Program.


Bacterial development & differentiation

The overall goal of our research is to unravel the mechanisms that allow bacteria to adapt and differentiate in response to changes in the environment. Bacteria have evolved at least three strategies that allow them to cope with such changes. One strategy centers on changes in gene expression ranging from changes in the expression of relatively few genes to changes in the expression of large numbers of genes culminating in cell differentiation. A second strategy centers on changes in the motility behavior of cells. Finally, a third strategy centers on changes to the cell cycle. To implement these strategies bacterial cells have to process vast amounts of information and then generate the appropriate output response. Information processing is carried out by complex networks of signal transduction proteins. A challenging problem in bacterial adaptation and differentiation is to understand how these protein networks are organized in space and time to allow the ordered execution of various tasks.

We use Myxococcus xanthus as a wonderful model system to study bacterial adaptation and differentiation. In particular, we study the signal transduction pathways and networks governing differentiation, motility and the cell cycle. In parallel approaches, we aim to understand how molecular machineries involved in motility and cell division function.


Viable Cell Counting

Plate counting is used to estimate the number of viable cells that are present in a sample.

Learning Objectives

Explain viable cell counting

Key Takeaways

Key Points

  • The spread plate relies on bacteria growing a colony on a nutrient medium so that the colony becomes visible to the naked eye and the number of colonies on a plate can be counted.
  • Selective media can be used to restrict the growth of non-target bacteria.
  • The pour plate method is used when the analysis is looking for bacterial species that grow poorly in air, for example water samples.

Key Terms

  • plate count: A means to identify the number of actively growing cells in a sample.

Viable Cell Counting

Selective media can be used to restrict the growth of non-target bacteria.: Urine cultured on Oxoid Brilliance UTI Agar plate. 1uL of urine spread onto the agar surface. The top sample is from patient with clinical urinary tract infection (UTI). The bottom sample is a mixed culture.

There are a variety of ways to enumerate the number of bacteria in a sample. A viable cell count allows one to identify the number of actively growing/dividing cells in a sample. The plate count method or spread plate relies on bacteria growing a colony on a nutrient medium. The colony becomes visible to the naked eye and the number of colonies on a plate can be counted. To be effective, the dilution of the original sample must be arranged so that on average between 30 and 300 colonies of the target bacterium are grown. Fewer than 30 colonies makes the interpretation statistically unsound and greater than 300 colonies often results in overlapping colonies and imprecision in the count. To ensure that an appropriate number of colonies will be generated several dilutions are normally cultured. The laboratory procedure involves making serial dilutions of the sample (1:10, 1:100, 1:1000 etc. ) in sterile water and cultivating these on nutrient agar in a dish that is sealed and incubated. Typical media include Plate count agar for a general count or MacConkey agar to count gram-negative bacteria such as E. coli. Typically one set of plates is incubated at 22°C and for 24 hours and a second set at 37°C for 24 hours. The composition of the nutrient usually includes reagents that resist the growth of non-target organisms and make the target organism easily identified, often by a color change in the medium. Some recent methods include a fluorescent agent so that counting of the colonies can be automated. At the end of the incubation period the colonies are counted by eye, a procedure that takes a few moments and does not require a microscope as the colonies are typically a few millimeters across.

The pour plate method is used when the analysis is looking for bacterial species that grow poorly in air. The initial analysis is done by mixing serial dilutions of the sample in liquid nutrient agar which is then poured into bottles. The bottles are then sealed and laid on their sides to produce a sloping agar surface. Colonies that develop in the body of the medium can be counted by eye after incubation. The total number of colonies is referred to as the Total Viable Count (TVC). The unit of measurement is cfu/ml (or colony forming units per milliliter) and relates to the original sample. Calculation of this is a multiple of the counted number of colonies multiplied by the dilution used. Examples of a viable cell count are spread plates from a serial dilution of a liquid culture and pour plates. With a spread plate one makes serial dilutions in liquid media and then spreads a known volume from the last tube in the dilution series. The colonies on the plate can then be counted and the concentration of bacteria in the original culture can be calculated. In the pour plate method a diluted bacterial sample is mixed with melted agar and then that mixture is poured into a petri dish. Again the colonies would be counted and the viable cell count calculated.


Colony Shape

It includes form, elevation, and margin of the bacterial colony.

Form of the bacterial colony: – The form refers to the shape of the colony. These forms represent the most common colony shapes you are likely to encounter. e.g. circular, irregular, filamentous, rhizoid, etc.

Elevation of the bacterial colony: It gives information about, how much does the colony rise above the agar. This describes the “side view” of a colony. These are the most common elevations e.g. flat, raised, umbonate (having a knobby protuberance), crateriform, convex, pulvinate (cushion-shaped).

Margin of bacterial colony: The margin or edge of a colony may be an important characteristic in identifying organisms. Common examples are entire (smooth), irregular, undulate (wavy), lobate, curled, filiform, etc.

Colonies that are irregular in shape and/or have irregular margins are likely to be motile organisms. Highly motile organisms swarmed over the culture media, such as Proteus spp.


Show/hide words to know

Differentiation: when a cell chooses a particular genetically determined path that causes it to perform only a few specialized tasks. more

DNA (deoxyribonucleic acid): molecular instructions that guide how all living things develop and function. more

Egg: a female gamete, which keeps all the parts of a cell after fusing with a sperm.

Gamete: specialized cells found in your reproductive organs that have half the amount of DNA of somatic cells. These cells combine to make a fertilized egg. more

Gene: a region of DNA that instructs the cell on how to build protein(s). As a human, you usually get a set of instructions from your mom and another set from your dad. more

Nucleus: where DNA stays in the cell, plural is nuclei.

Organism: a living thing that can be small like bacteria or large like an elephant.

Somatic cells: the cells in your body, except for gametes. Soma is Latin for body.

Sperm: a male gamete, which only transfers its DNA to the egg. more


Pathophysiology

Staphylococcus aureus is a gram-positive, catalase-positive, coagulase-positive cocci in clusters. S. aureus canꃊuse inflammatory diseases, including skin infections, pneumonia, endocarditis, septic arthritis, osteomyelitis, and abscesses. S. aureus can also cause toxic shock syndrome (TSST-1), scalded skin syndrome (exfoliative toxin, and food poisoning (enterotoxin). 

Staphylococcus epidermidis is a gram-positive, catalase-positive, coagulase-negative cocci in clusters and is novobiocin sensitive. S. epidermidis commonly infects prosthetic devices and IV catheters producing biofilms. Staphylococcus saprophyticus is novobiocin resistant and is a normal flora of the genital tract and perineum. S. saprophyticus򠫌ounts for the second most common cause of uncomplicated urinary tract infection (UTI). 

Streptococcus pneumoniae is a gram-positive, encapsulated, lancet-shaped diplococci, most commonly causing otitis media, pneumonia, sinusitis, and meningitis. Streptococcus viridans਌onsist of Strep. mutansਊnd Strep mitis found in the normal flora of the oropharynx commonly cause dental carries and subacute bacterial endocarditis (Strep. sanguinis).

Streptococcus pyogenes is a gram-positive group A cocci that can cause pyogenic infections (pharyngitis, cellulitis, impetigo, erysipelas), toxigenic infections (scarlet fever, necrotizing fasciitis), and immunologic infections (glomerulonephritis and rheumatic fever). ASO titer detects S. pyogenes infections.

Streptococcus agalactiae is a gram-positive group B cocci that colonize the vagina and is found mainly in babies. Pregnant women need screeningਏor Group-B Strep (GBS) at 35 to 37 weeks of gestation. 

Enterococci is a gram-positive group D cocci found mainly in the colonic flora and can cause biliary tract infections and UTIs. Vancomycin-resistant਎nterococci (VRE) are an important cause of nosocomial infections. 

Clostridia is a gram-positive spore-forming rod consisting of C. tetani, C. botulinum, C. perfringens, and C. difficile. C. difficile is often secondary to antibiotic use (clindamycin/ampicillin), PPI use, and recent hospitalization. Treatment involves primarily with oral vancomycin.

Bacillus anthracis is a gram-positive spore-forming rod that produces anthrax toxin resulting in an ulcer with a black eschar. Bacillus cereus is a gram-positive rod that can be acquired from spores surviving under-cooked or reheated rice. Symptoms include nausea, vomiting, and watery non-bloody diarrhea. 

Corynebacterium diphtheria is a gram-positive਌lub-shaped rod that can cause pseudomembranous pharyngitis, myocarditis, and arrhythmias. Toxoid vaccines prevent diphtheria.

Listeria monocytogenes is a gram-positive rod acquired by the ingestion of cold deli meats and unpasteurized dairy products or by vaginal transmission during birth. Listeria can cause neonatal meningitis, meningitis in immunocompromised patients, gastroenteritis, and septicemia. Treatment includes ampicillin. 



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