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CLINICAL BACTERIOLOGY LECTURE | Prof. Ethel Mangada | transcribed by: Joie, Maj, Rish Unit 3: GENERAL CHARACTERISTIC OF BACTERIA...

CLINICAL BACTERIOLOGY LECTURE | Prof. Ethel Mangada | transcribed by: Joie, Maj, Rish Unit 3: GENERAL CHARACTERISTIC OF BACTERIA MORPHOLOGY ➔ Vary in size, morphology and cell-to-cell arrangement OUTLINE and in the chemical composition and structure of the I Prokaryotic Vs. Eukaryotic cell wall A Eukaryotic ➔ Bacterial cell wall differences provide the basis for the B Prokaryotic Gram stain II Bacteria A Morphology A. BACTERIAL SIZE B Cell Structure Most clinically relevant bacterial species range in size from 0.25 t0 1um in width and 1 to 3um in light PROKARYOTIC VS. EUKARYOTIC CELLS Bacterium in some hundred-fold larger than a virus and ten-fold smaller than a eukaryotic cells Chemically similar ○ Both contain nucleic acids, protein, lipid and carbohydrate B. BACTERIAL SHAPE Use the same kinds of chemical reaction to Common bacterial cellular morphologies include metabolize food, build protein and store energy ○ Cocci: Circular Difference: Structure of cell walls and membranes, ○ Coccobacilli:Ovoid and he absence of organelles (specialized cellular ○ Bacillus: rod shaped structure that have specific function) ○ Uniform tapered, pointed ends ○ Curved EUKARYOTES ○ Spiral: helical like corkscrew. Spirochetes vary in length and in the number of helical ➔ DNA is found in the cell’s nucleus, which is separated turns (not all helical bacteria are called from the cytoplasm by a nuclear membrane and the spirochetes DNA is found in multiple chromosomes ○ Pleomorphic: no defined shape ➔ DNA is consistently associated with chromosomal protein called histones and nonhistones C. BACTERIAL ARRANGEMENT ➔ Have a number of membrane-enclosed organelles ➔ Cell walls, when present re chemically simple Pairs ➔ Cells division usually involves MITOSIS Chains Grape-like clusters Group of four PROKARYOTES Packet of eight ➔ DNA is not enclosed within a membrane and usually Palisades singular circularly arranged chromosome Chinese Characters ➔ DNA is not associated with histones: other proteins are associated with the DNA ➔ Lack membrane-enclosed organelles CELL STRUCTURE ➔ Cell walls almost always contain the complex polysaccharide peptidoglycan ➔ Usually divide by BINARY FISSION ◆ DNA is copied, and the cell splits into two CELL ENVELOPE cells ◆ Involved fewer structures and processes outermost structure, comprises: then eukaryotic cell division A. Outer Membrane ➔ in gram-negative bacteria only BACTERIA organism that lack a nuclear membrane and true B. Cell Wall nucleus ➔ composed of the peptidoglycan macromolecule Classified as prokaryotes (Greek: before kernel- (murein layer) nucleus) having no mitochondria, endoplasmic reticulum (ER) or Golgi bodies C. Periplasm ➔ in gram-negative bacteria only D. Cytoplasmic or Cell Membrane ➔ encloses the cytoplasm "DO NOT BE ANXIOUS ABOUT ANYTHING, BUT IN EVERY SITUATION, BY PRAYER AND PETITION, WITH THANKSGIVING, PRESENT YOUR REQUESTS TO GOD. AND 1 THE PEACE OF GOD, WHICH TRANSCENDS ALL UNDERSTANDING, WILL GUARD YOUR HEARTS AND YOUR MINDS IN CHRIST JESUS."  CLINICAL BACTERIOLOGY LECTURE | Prof. Ethel Mangada | transcribed by: Joie, Maj, Rish Unit 3: GENERAL CHARACTERISTIC OF BACTERIA ○ Polymers of these subunits across-link to one another by means of peptide bridges to form peptidoglycan sheets OUTER MEMBRANE ○ Layers of these sheets are crosslinked with one another, forming multilayered, ➔ found only in gram-negative bacteria cross-linked structure of considerable ➔ function as the cell’s initial barrier to the environment strength ➔ serve as primary permeability barriers to hydrophilic ○ Referred to as the murein sacculus, or sack and hydrophobic compounds and contain essential this peptidoglycan structure surrounds the enzymes and other proteins located in the periplasmic entire cells space ➔ bilayered structure composed of lipopolysaccharide NOTES → gives the surface of gram- negative bacteria a net negative charge Different types of cell wall structure traditionally ave bee ➔ plays a significant role in the ability of certain bacteria categorized according to their staining characteristics to cause disease Major type of cell walls: gram-positive and gram-negative types Porins Mycobacteria: stain gram-positive, have a modified cell ○ protein structures scattered throughout the wall called an ACID-FAST CELL WALL lipopolysaccharide macromolecules Mycoplasmas: microorganism that have no cell wall ○ water-filled structures that control the passage of nutrients and other solutes, including antibiotics, through the outer membrane ○ GRAM-POSITIVE CELL WALL ○ number and types of porins vary with ○ Composed of a very thick protective bacterial species peptidoglycan murein layer ○ ○ Consist of glycan (polysaccharide) chain of ○ influence the extent to which various alternating N-acetyl-D-glucosamine (NAG) substances pass through the outer and N-acetyl-d-muramic acid (NAM) membranes of different bacteria ○ Many antibiotics effective against gram-positive organism (e.g penicillin) ac by Murein Lipoproteins preventing synthesis of peptidoglycan ○ facilitate the attachment of the outer membrane to the next internal layer in the NOTES cell envelope, the cell wall Gram-negative bacteria: thinner layer of peptidoglycan and a different cell wall structure, are less affected by these antibiotics CELL WALL (MUREIN LAYER) Referred to as the peptidoglycan or murein layer Other components of the gram-positive cell wall that Gives the bacterial cell shape and strength to penetrate to the exterior of the cell are: withstand changes in environmental osmotic pressure that would otherwise result in cell lysis TEICHOIC ACID Protects against mechanical disruption of the cell and ○ Anchored to the peptidoglycan offers some Barrie to the passage of larger (N-acetylmuramic acid) substances ○ glycerol or ribitol phosphate polymers Synthesis and structure are often the primary targets combined with for the development and design of several ○ various sugars, amino acids, and amino antimicrobial agents sugars Structure is composed of disaccharide-pentapeptide subunits LIPOTEICHOIC ACID ○ anchored to the PM N-acetyl-D-glucosamine and N-acetyl-D-muramic ○ linked to the next underlying layer, PM or acid cellular ○ Salternating sugar components (moieties), ○ membrane with the amino acid chain linked to ○ These two components are unique to the N-acetylmuramic acid molecules gram-positive cell wall "DO NOT BE ANXIOUS ABOUT ANYTHING, BUT IN EVERY SITUATION, BY PRAYER AND PETITION, WITH THANKSGIVING, PRESENT YOUR REQUESTS TO GOD. AND 2 THE PEACE OF GOD, WHICH TRANSCENDS ALL UNDERSTANDING, WILL GUARD YOUR HEARTS AND YOUR MINDS IN CHRIST JESUS."  CLINICAL BACTERIOLOGY LECTURE | Prof. Ethel Mangada | transcribed by: Joie, Maj, Rish Unit 3: GENERAL CHARACTERISTIC OF BACTERIA ○ Other antigenic polysaccharides may be present on the surface of the peptidoglycan ACID-FAST CELL WALL layer →have a gram-positive cell wall structure →contain a waxy layer of glycolipids and fatty acids TEICHURONIC ACIDS (mycolic acid) bound to the exterior of the cell wall ○ similar polymers, but the repeat units include →More than 60% of the cell wall is lipid sugar ○ acids (eg, N-acetylmannosuronic or Mycolic acid d-glucosuronic acid) → major lipid component ○ instead of phosphoric acids →strong “hydrophobic” molecule that forms a lipid shell ○ synthesized in place of teichoic acids when around the organism and affects its permeability ○ phosphate is limiting →makes Mycobacterium spp. difficult to stain with the Gram stain GRAM-NEGATIVE CELL WALL NOTE!!! ➔ composed of two layers: Mycobacterium and Nocardia ➔ Inner peptidoglycan layer →stain a faint blue (gram-positive) color ◆ much thinner than in gram-positive cell walls →best stained with an acid-fast stain ➔ Outer membrane ◆ outside the peptidoglycan layer is and ABSENCE OF CELL WALL additional outer membrane lack a cell wall and contain STEROLS in their cell ◆ contains proteins, phospholipids, and membranes lipopolysaccharide (LPS) →lack the rigidity of the cell wall →seen in various shapes microscopically LPS contains three regions: Example: Mycoplasma and Ureaplasma a. O–specific polysaccharide= antigenic b. Core polysaccharide= ketodeoxyoctanoic **Gram-positive and gram-negative cells can lose their cell acid (KDO) and heptose walls and grow as L-forms in media supplemented with c. cLipid A (also called endotoxin)= inner, major serum or sugar to prevent osmotic rupture of the cell constituents membrane LPS Functions: PERIPLASMIC SPACE ○ Vital in evading the host defenses ○ Contribute to the negative charge of the typically found only in gram-negative bacteria ○ bacterial surface, which stabilizes the bounded by the internal surface of the outer ○ membrane structure membrane and the external surface of the cellular. ○ Considered as an endotoxin membrane encompassing the thin peptidoglycan layer contains the murein layer, consisting of a gel-like ONGOING CONSTRUCTION matrix containing nutrient-binding proteins that assist in the capture of nutrients from the environment. Lipid A moiety contains several enzymes involved in the degradation →consists of phosphorylated glucosamine disaccharide of macromolecules and detoxification of units to which are attached a number of long-chain environmental solutes, including antibiotics that enter fatty acids through the outer membrane →responsible for producing fever and shock conditions in patients infected with gram-negative bacteria NOTE! Periplasmic space is absent in gram-positive bacteria Outer membrane function: ✓Acts as a barrier to hydrophobic compounds and harmful CYTOPLASMIC (INNER) MEMBRANE substances ✓ Acts as a sieve, allowing water-soluble molecules to enter present in both gram-positive and gram-negative through protein-lined channels called porins bacteria and is the deepest layer of the cell envelope ✓Provides attachment sites that enhance attachment to host consist of phospholipid bilayer, various proteins cells (70%), including a number of enzymes vital to cellular ✓Strong negative charge is an important factor in evading metabolism phagocytosis serves as an additional osmotic barrier ✓Acts as a barriers to toxic substances that prevents Absence of sterols movement inside the cell "DO NOT BE ANXIOUS ABOUT ANYTHING, BUT IN EVERY SITUATION, BY PRAYER AND PETITION, WITH THANKSGIVING, PRESENT YOUR REQUESTS TO GOD. AND 3 THE PEACE OF GOD, WHICH TRANSCENDS ALL UNDERSTANDING, WILL GUARD YOUR HEARTS AND YOUR MINDS IN CHRIST JESUS."  CLINICAL BACTERIOLOGY LECTURE | Prof. Ethel Mangada | transcribed by: Joie, Maj, Rish Unit 3: GENERAL CHARACTERISTIC OF BACTERIA Exceptions: Mycoplasma→ incorporate sterols (e.g., → particularly common in aerobic organisms, which have an cholesterol), into their membranes when growing in easier time generating an ion-motive force than do anaerobes sterol-containing media FUNCTIONS: ✓Transport of solutes into and out of the cell ✓Housing of enzymes involved in outer membrane synthesis, Three basic types: cell wall synthesis, and the assembly and secretion of extracytoplasmic and extracellular substances Uniport → catalyze the transport of a substrate independent of any ✓Generation of chemical energy (i.e., ATP) coupled ion ✓Cell motility Symport → simultaneous transport of two substrates in the same ✓Mediation of chromosomal segregation during replication direction by a single carrier ✓Housing of molecular sensors that monitor chemical and Antiport physical changes in the environment → simultaneous transport of two like-charged compounds in opposite directions by a common carrier (40% of the ✓Electron transport and oxidative phosphorylation in aerobic substrates transported by E coli) species b. ABC transport ✓Excretion of hydrolytic exoenzymes → uses ATP directly to transport solutes into the cell Gram-negative: transport of many nutrients is facilitated by specific binding proteins located in the periplasmic space PERMEABILITY AND TRANSPORT Gram-positive: binding proteins are attached to the outer surface of the cell membrane PASSIVE TRANSPORT GROUP TRANSLOCATION - relies on diffusion, uses no energy, and operates only when the solute is at higher concentration outside than inside the cell → vectorial metabolism → not active transport because no concentration gradient is a. Simple diffusion involved → accounts for the entry of very few nutrients, including → allows bacteria to use their energy resources efficiently by dissolved oxygen, carbon dioxide, and water itself coupling transport with metabolism b. Facilitated diffusion → selective and uses no energy so the solute never achieves SPECIAL TRANSPORT PROCESSES an internal concentration greater than what exists outside the cell (e.g., Glycerol) Siderophores c. Channel proteins - compounds that chelate Fe and promote its transport → form selective channels that facilitate the passage of as a soluble complex specific molecules ✓ Some pathogenic bacteria use specific receptors that bind host transferrin and lactoferrin (as well as other iron-containing host proteins) ACTIVE TRANSPORT a. Ion-coupled transport → move a molecule across the cell membrane at the expense of a previously established ion gradient such as protonmotive or sodiummotive force "DO NOT BE ANXIOUS ABOUT ANYTHING, BUT IN EVERY SITUATION, BY PRAYER AND PETITION, WITH THANKSGIVING, PRESENT YOUR REQUESTS TO GOD. AND 4 THE PEACE OF GOD, WHICH TRANSCENDS ALL UNDERSTANDING, WILL GUARD YOUR HEARTS AND YOUR MINDS IN CHRIST JESUS."  CLINICAL BACTERIOLOGY LECTURE | Prof. Ethel Mangada | transcribed by: Joie, Maj, Rish Unit 3: GENERAL CHARACTERISTIC OF BACTERIA CYTOPLASMIC STRUCTURE Small Plasmid - resistant to tetracyclines and chloramphenicol a. Ribosomes - site of protein biosynthesis and gives the cytoplasm a granular structure INCLUSIONS BODIES - Consist of RNA and proteins - 70S in size and separates into two subunits, 50S and 30S - Serve as the energy source or food reserve of the bacteria or as a reservoir of structural building blocks NOTE!!! - Composed mainly of polysaccharides, they lessen osmotic pressure Streptomycin and Gentamicin Examples: glycogen, cyanophysin → attach to the 30S subunit and interfere with protein granules,poly-B-hydroxybutyrate granules, carboxysomes synthesis (cyanobacteria, nitrifying bacteria and thiobacilli), gas vacuoles (cyanobacteria, halobacterium and thiothrix) and Erythromycin and Chloramphenicol polyphosphate granules(volutin and metachromatic granules → interfere with protein synthesis by attaching to the 50S subunit Two Common Types of Granules: a. Glycogen b. Genome - storage form of glucose - Consist of a single, circular chromosome b. Polyphosphate Granules - lacks nuclear membrane and mitotic apparatus - storage form for inorganic phosphates - Appears as diffused nucleoid or chromatin body that - Source of phosphate for nucleic acid and is attached to a mesosome (sac-like structure) phospholipid sythesis Examples: Babes-Ernst Bodies (C. diptheria) Nucleoid = Feulgen positive Bipolar Bodies (Y. pestis) Much Granules (M. tuberculosis) - Consists of a single continuous circular molecule ranging in size from 0.58 to almost 10 million base Poly-B-hydroxybutyric acid (PHB) pair - Lipid like compound consisting of chains of B- hydroxybutyric acid units connected through ester Exceptions: Borrelia burgdorferi and Streptomyces linkages coelicolor - Produced when the source of nitrogen, sulfur or phosphorus is limited and there is excess carbon in - Few bacteria have dissimilar chromosomes: Vibrio the medium cholera and Brucella melitensis PHB and Glycogen - carbon source when protein and nucleic acid c. Plasmid synthesis are resume - extrachromosomal, double-stranded element of DNA that is associated with virulence Sulfur Granules - Located in the cytoplasm and serve as a site for the - hydrogen sulfide and thiosulfate genes to code for antibiotic resistance and toxin production - Not essential for bacterial growth so a bacterial cell ENDOSPORES/ ASEXUAL SPORES may or may not contain a plasmid - Sometimes disappears during cell division and it can make bacteria (mostly Gram-neg) pathogenic Small, dormant structures located inside the bacterial cell →Aid in the survival of bacteria against external conditions Two Kinds of Plasmid →Produced within vegetative cells of some Gram-pos bacteria →Composed of dipicolinic acid and calcium ions: CALCIUM Large Plasmid DIPICOLINATE - responsible for the production of B-lactamase that →Some locations could be a means of microscopically provide resistance to B-lactam antibiotics (penicillin identifying and oxacillin) bacteria "DO NOT BE ANXIOUS ABOUT ANYTHING, BUT IN EVERY SITUATION, BY PRAYER AND PETITION, WITH THANKSGIVING, PRESENT YOUR REQUESTS TO GOD. AND 5 THE PEACE OF GOD, WHICH TRANSCENDS ALL UNDERSTANDING, WILL GUARD YOUR HEARTS AND YOUR MINDS IN CHRIST JESUS."  CLINICAL BACTERIOLOGY LECTURE | Prof. Ethel Mangada | transcribed by: Joie, Maj, Rish Unit 3: GENERAL CHARACTERISTIC OF BACTERIA →Responsible for perpetuation, but not muliplication Exception: Poly-D-glutamic acid capsules of Bacillus Examples: Bacillus and Clostridium anthracis and Bacillus licheniformis Types of spores according to location: - Protects the bacteria(virulence factor) from the a. Terminal spore= Clostridium tetani attacks of human defense system since it resist b. Subterminal spore= Clostridium botulinum phagocytosis and desiccation c. Central spore= Bacillus anthracis - capsules sometimes must be removed to detect the somatic (cell wall) antigens present Properties of Endospores - Capsule removal is accomplished by boiling a 1. Core→ spore protoplast suspension of the microorganism →contains a complete nucleus (chromosome), all of the - Does not ordinarily stain with use of common components of the protein-synthesizing apparatus, and laboratory stains, such as Gram or India ink→ an energy-generating system based on glycolysis appears as a clear area (“halo”-like) 2. Spore wall→ innermost layer surrounding the inner spore membrane b. Slime Layer →contains normal peptidoglycan and becomes the cell - Unorganized material that is loosely attached to the wall of the germinating vegetative cell cell wall 3. Cortex →thickest layer of the spore envelope - Made up of polysaccharide → contains an unusual type of peptidoglycan, with - Can either inhibit phagocytosis or aid in the many fewer cross-links than are found in cell wall adherence of the bacteria to the host tissue or peptidoglycan synthetic implants - facilitates and maintains bacterial colonization of Coat→ composed of a keratin-like protein containing biologic (e.g., teeth) and inanimate (e.g., prosthetic many intramolecular disulfide bonds heart valves) surfaces through the formation of → Impermeability of this layer confers on spores their biofilms relative resistance to antibacterial chemical agents 5. Exosporium→ composed of proteins, lipids, and Extracellular Polymeric Substance (EPS) carbohydrates - helps cells in a biofilm attach to their target →consists of a paracrystalline basal layer environment and to each other and - protects the cells within it, facilitates communication a among them, and enables the cells to survive by hairlike outer region attaching to various surfaces in their natural Example: B. anthracis and B. cereus environment FLAGELLA CELLULAR APPENDAGES - exterior protein filaments that rotate and cause bacteria to be motile - play a role in the mediation of infection and in laboratory identification, varies among bacterial - complex structures, mostly composed of the protein species and even among strains within the same flagellin, intricately embedded in the cell envelope species - thread-like appendages composed entirely of protein, 12–30 nm in diameter GLYCOCALYX - plays an important role in survival and the ability of - Outward complex of polysaccharide on the bacterial certain bacteria to cause disease surface and other cells - Helps the bacteria to attach to the surface of the solid - antigenic (H antigens), and some of the immune objects or tissues responses to infection are directed against these - Appears as a capsule or a slime layer proteins a. Capsule - Gliding motility: Capnocytophaga, Cyanobacteria, - organized and is firmly attached to the cell wall Myxobacteria, - immediately exterior to the murein layer of gram- positive bacteria and the outer membrane of gram- Flagellum is attached to the bacterial cell body by a complex negative bacteria structure consisting: - Made up of polysaccharide polymers "DO NOT BE ANXIOUS ABOUT ANYTHING, BUT IN EVERY SITUATION, BY PRAYER AND PETITION, WITH THANKSGIVING, PRESENT YOUR REQUESTS TO GOD. AND 6 THE PEACE OF GOD, WHICH TRANSCENDS ALL UNDERSTANDING, WILL GUARD YOUR HEARTS AND YOUR MINDS IN CHRIST JESUS."  CLINICAL BACTERIOLOGY LECTURE | Prof. Ethel Mangada | transcribed by: Joie, Maj, Rish Unit 3: GENERAL CHARACTERISTIC OF BACTERIA SPIROCHETES → group of bacteria that have unique structure and motility → move by means of AXIAL FILAMENTS OR Hook ENDOFLAGELLA - short curved structure that appears to act as the universal joint between the motor in the basal True motility and Brownian Movement are best observed structure and the flagellum through the HANGING DROP METHOD Basal body True motility→ bacteria seem to be going in a definite - bears a set of rings, one pair in gram- positive direction bacteria and two pairs in gram-negative bacteria Brownian movement→ bacteria bounce back and forth Filament rapidly due to the bombardment of molecules of water → long outermost region Taxis → constant in diameter and contains the globular (roughly - movement of bacteria toward or away from a spherical) protein flagellin arranged in several chains that particular stimulus intertwine and form a helix around a hollow core Ways of Demonstrating Motility In the Lab: Motility ✓Hanging Drop Method - ability of an organism to move by itself ✓SIM ✓Flagellar staining “Run" or “Swim” ✓Serologic test - bacterium moves in one direction for a length of time ✓Fluorescent Antibody Technique(FAT) ✓Swarming Phenomenon "Runs“ ✓Darkfield Microscopy - interrupted by periodic, abrupt, random changes in direction called "tumbles" , then, a "run" resumes "Tumbles“ PILI (FIMBRIA) - caused by a reversal of flagellar rotation - hairlike, proteinaceous structures that extend from the cell membrane into the external environment; some Proteus may be up to 2 μm long - endowed with many flagella - can "swarm," or show rapid wavelike movement - Hair-like microfibrils usually produced by flagellated across a solid culture medium Gram-negative bacteria observable by electron microscopy Arrangement of the Flagella: a. Atrichous→ without flagellum - serve as adhesins that help bacteria attach to animal b. Monotrichous→ single flagellum at one end host cell surfaces, often as the first step in c. Amphitrichous→ single flagellum at both ends establishing infection d. Lophotrichous→ tuff or group of flagella on one end or both ends - composed of structural protein subunits--pilins e. Peritrichous→ entire cell surface covered with flagella Twitching motility - a pilus extends by the addition of subunits of pilin, Axial Filaments makes contact with a surface or another cell, and then → bundles of fibrils that arise at the ends of the cell beneath an retracts (powerstroke) as the pilin subunits are outer sheath and spiral around the cell disassembled---grappling hook model →anchored at one end of the spirochete →have a structure similar to that of flagella - Results in short, jerky, intermittent movements →Rotation of the filaments produces a movement of the outer Example: Pseudomonas aeruginosa, Neisseria sheath that propels the spirochetes in a spiral motion gonorrhoeae, and some strains of E. coli →Movement is similar to the way a corkscrew moves through a cork Common Pili or Ordinary Pili - Play a role in bacterial adherence to surfaces thus contributing to virulence Note!!! "DO NOT BE ANXIOUS ABOUT ANYTHING, BUT IN EVERY SITUATION, BY PRAYER AND PETITION, WITH THANKSGIVING, PRESENT YOUR REQUESTS TO GOD. AND 7 THE PEACE OF GOD, WHICH TRANSCENDS ALL UNDERSTANDING, WILL GUARD YOUR HEARTS AND YOUR MINDS IN CHRIST JESUS."  CLINICAL BACTERIOLOGY LECTURE | Prof. Ethel Mangada | transcribed by: Joie, Maj, Rish Unit 3: GENERAL CHARACTERISTIC OF BACTERIA Sex Pilus - serves as the conduit for the passage of DNA from donor to recipient during conjugation - present only in cells that produce a protein referred to as the F factor F-positive cells initiate conjugation only with F-negative cells, thereby limiting the conjugative process to cells capable of transporting genetic material through the hollow sex pilus NOTE!!! Streptococci - fimbriae are the site of the main surface antigen---M protein Lipoteichoic acid, associated with these fimbriae, - responsible for the adherence of group A streptococci to epithelial cells of their hosts N. gonorrhoeae - able to make pili of different antigenic types (antigenic variation) "DO NOT BE ANXIOUS ABOUT ANYTHING, BUT IN EVERY SITUATION, BY PRAYER AND PETITION, WITH THANKSGIVING, PRESENT YOUR REQUESTS TO GOD. AND 8 THE PEACE OF GOD, WHICH TRANSCENDS ALL UNDERSTANDING, WILL GUARD YOUR HEARTS AND YOUR MINDS IN CHRIST JESUS."