GEN MICRO IMMUNHOST
GEN MICRO IMMUNHOST BIO 226N
Popular in Course
Popular in Biology
This 23 page Class Notes was uploaded by Ezequiel Orn on Sunday September 6, 2015. The Class Notes belongs to BIO 226N at University of Texas at Austin taught by Staff in Fall. Since its upload, it has received 42 views. For similar materials see /class/181732/bio-226n-university-of-texas-at-austin in Biology at University of Texas at Austin.
Reviews for GEN MICRO IMMUNHOST
Report this Material
What is Karma?
Karma is the currency of StudySoup.
You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!
Date Created: 09/06/15
Molecular Microbiology 2003 493 571 580 Micro Review Bacterial shape Kevin D Young Department of Microbiology and Immunology University of North Dakota School of Medicine Grand Forks ND 582029037 USA Architecture is the adaptation of form to resist force John Ruskin 1874 Summary In freeliving eubacteria an external shell of pepti doglycan opposes internal hydrostatic pressure and prevents membrane rupture and death At the same time this wall imposes on each cell a shape Because shape is both stable and heritable as is the ability of many organisms to execute defined morphological transformations cells must actively choose from among a large repertoire of available shapes How they do so has been debated for decades but recently experiment has begun to catch up with theory Two discoveries are particularly informative First specific protein assemblies nucleated by FtsZ MreB or Mbl appear to act as internal scaffolds that influence cell shape perhaps by correctly localizing synthetic enzymes Second defects in cell shape are correlated with the presence of inappropriately placed metabol ically inert patches of peptidoglycan When combined with what we know about mutants affecting cellular morphology these observations suggest that bacte ria may fabricate specific shapes by directing the syn thesis of two kinds of cell wall a longlived rigid framework that defines overall topology and a meta bolically plastic peptidoglycan whose shape is directed by internal scaffolds Introduction At present we cannot explain in comfortable detail how even one bacterium constructs itself much less how the vast constellation of prokaryotic shapes arises Our igno rance is broad and profound and encompasses the most basic of questions For example out of the universe of Accepted 1 May 2003 For correspondence Email kyoungmedicinenodakedu Tel 1 701 777 2624 Fax 1 701 777 2054 2003 Blackwell Publishing Ltd doi10104613652958200303607X possibilities how do cells select create and maintain a defined size length and width Why are the dimensions of most cells uniform instead of irregular How do unevenly shaped cells manage to endow their descen dents with reproducible irregularities Why do some cells branch profusely and others not at all And how do cells preserve or adjust these parameters during growth or when they enter and exit different environments or as they progress through a developmental cycle Unfortunately especially when compared with advances in other physiological arenas the answers to these questions have changed relatively little in over 30 years since the incisive reviews of Henning and Schwarz who charted the broad outlines of the problems and their solutions Henning and Schwarz 1973 Hen ning 1975 Subsequent treatments reemphasized the problems and illuminated pieces of the puzzle Harold 1990 Nanninga 1998 but despite a more thorough knowledge of peptidoglycan synthesis and bacterial cell division our understanding of morphological control remains dim and untidy Happily recent discoveries are fleshing out previously vague ideas and substantial progress is being made in identifying the molecular mech anisms responsible for building cells of diverse but indi vidual shapes Scaffolds In eubacteria the loadbearing structure is peptidoglycan an interlaced monomolecular meshwcrk that defines cell shape Holtje 1998 Nanninga 1998 Because the chemical composition of peptidoglycan is similar among cells of diverse shapes individual building blocks cannot explicitly define the form of the wall Schwarz and Leutgeb 1971 de Pedro etal 2003 But if the shape of the prokaryotic cell wall does not appear spontaneously then its form must be directed and for over 40 years the question has been By what As in any construction project there are only two general ways to define the boundaries of a wall by internal or external scaffolding Internal scaffolding Consider the problem of setting the extent and unifor mity of a cell s diameter There are three ways to do this 572 K D Young Fig 1 Faithful replication of a series of hoops having defined radii could define the boundaries of the side walls in a rodshaped organism Fig 1 top or a spiral of defined pitch could accomplish the same effect Fig 1 middle However to set the diameter de novo by either of these two mechanisms would require a chemically determined selfassembly of the hoop or spi ral components If such selfdirected assembly does not occur then preexisting structures must act as templates to dictate the diameter of the replicas The third way to inscribe a structure of defined radius is to use a com pass a device with one stationary end attached to another that swings freely to describe the circumference of a circle As a biological example imagine molecular spokes of defined and equal length attached to a cen tral linear polymer Fig 1 bottom If the distal ends of the spokes were embedded in the cytoplasmic mem brane osmotic pressure would extend the membrane to the limits of the spokes creating a rodshaped organism Fig 1 bottom Interestingly prokaryotes seem to use all three mecha nisms The FtsZ protein is the earliest known component of a ring hoop required for septation Buddelmeijer and Beckwith 2002 Lutkenhaus 2002 However the diame Spokes Model Fig 1 Models of possible shapedefining scaffolds Each schematic of a rodshaped bacterium contains one of three different internal structures Cell diameter is determined by one or more hoops of defined radius top by a spiral of defined pitch centre or by an axleandspoke arrangement bottom ter of this structure is not set by selfassembly because the Z ring contracts during cell division and incomplete arcs can initiate septation Addinall and Lutkenhaus 1996 Pas elal 2001 de Pedro elal 2001 Also FtsZ polymerizes in vitro as straight filaments flat sheets or tiny rings Lutkenhaus and Addinall 1997 Lu elal 2000 Thus the Z ring does not define an independent radius though it may help retain or reproduce an existing diameter Potential spiral scaffolds are constructed by the MreB and Mbl proteins of Bacillus sublilis and the MreB homo logue in Escherichia coli which assemble on the cyto plasmic face of the inner membrane Wachi elal 1987 Jones elal 2001 CarballidoLopez and Errington 2003 Mutations in these proteins cause bacteria to grow as abnormal spheroids or misshapen rods indicat ing the proteins regulate cell shape Abhayawardhane and Stewart 1995 Jones elal 2001 The diameters of these spirals are also not selfgenerated the helical pitch of Mbl polymers changes with cell length and protein concentration CarballidoLopez and Errington 2003 and MreB filaments created in vitro have variable curva tures that differ from the innate diameter of the bacteria from which the proteins originate van den Ent elal 2001 Therefore these filaments like the Z ring proba bly assemble along the inner surface of preexisting membrane CarballidoLopez and Errington 2003 FtsZ itself may behave as a transient spiral scaffold because evanescent Z spirals appear at the end of septation and overproduction of FtsZGFP produces longlived helices Lutkenhaus and Addinall 1997 Sun and Margolin 2001 Lutkenhaus 2002 As for the third mechanism so far the axleandspoke structure has been observed in only one organism Myco plasma pneumoniae which has no peptidoglycan or other external cell wall to maintain its unique shape This microbe has no MreBlike internal scaffold Jones elal 2001 Instead ultrastructural analysis reveals that the rodshaped extension of M pneumoniae appears to be defined and supported by an internal bladelike rod with spokes attached to a wheellike complex Hegermann elal 2002 conceptually like that illustrated in Fig1 bottom Osmotic pressure as internal scaffold One oftendiscussed fourth alternative is that turgor pressure created by osmotic force dictates cell shape obviating the need for any more complex machinery The Surface Stress Theory Koch 1988 proposes that the cytoplasmic membrane behaves as an internal scaf fold actually a fully formed mould the shape of which is determined by nothing more than osmotic pressure and two stable poles of defined diameter Thus cell 2003 Blackwell Publishing Ltd Molecular Microbiology 49 571 580 shape is just a matter of synthesizing peptidoglycan onto the outer surface of the membrane to form a rigid wall Although the physics of turgor pressure are important and must influence the way cells and enzymes operate Koch 1988 2000 the Surface Stress Theory has three major problems in trying to explain cell shape First the theory assumes the existence of cellular structures inert poles of defined diameter whose mechanism of synthe sis is not generated within the theory itself leaving unad dressed a major determinant of cell shape Second the theory is at odds with the behaviour of mutants past and present whose shape changes dramatically without altering either the poles or internal pressure In particu lar recent appreciation of the roles of the MreB and Mbl proteins indicates that cells play a more active role in shape determination than envisioned by the original theory Finally and most telling the theory does not account for the spectacular diversity of prokaryotic shapes and sizes Although osmotic pressure must play a critical role we must look elsewhere for the specific mechanisms by which a cell adapts its form in response to this force External scaffolding In principle shapedefining hoops or spirals could be placed just as easily outside the cytoplasmic membrane In fact peptidoglycan is often envisioned as arranged as a series of parallel hoops whose precise replication deter mines and maintains a cell s diameter Holtje 1998 Unfortunately there is no direct experimental proof for such hoops lnstead according to current data individual glycan polymers are so short that to encircle E collonce would require 200 300 interconnected chains Holtje 1998 raising the question of how a specific diameter could be defined or maintained Another possibility is that peptidoglycan might be roughly spiral Mendelson 1982 a concept which may gain new support with recent spec ulations about the role of Mbl helices in peptidoglycan synthesis CarballidoLopez and Errington 2003 How ever even if true such helical peptidoglycan would owe its existence to an internal scaffold instead of being an independent determinant of cell shape Another possible external scaffold in Gram negative eubacteria is the outer membrane In E coll certain lipo proteins link peptidoglycan to this membrane and the strength of this association might contribute to the main tenance of cell length Cooper 1991 Such a relationship is difficult to prove but mutation or inactivation of several outer membrane components does alter cell shape For example an ompAlpp double mutant and some lpp mutants are spherical Sonntag elal 1978 Hiemstra elal 1987 and overexpression of the Nlpl lipoprotein 2003 Blackwell Publishing Ltd Molecular Microbiology 49 571 580 Bacterial shape 573 induces cells to form prolate ellipsoids whose poles are more pointed than normal Ohara elal 1999 Also deg radation of the OmpA protein by neutrophils destroys the uniform shape of E coll creating cells that appear as irregularly shaped flattened discs Belaaouaj elal 2000 Clearly the outer membrane or its proteins affect cell shape However we cannot say whether the effects arise from the disruption of an external scaffold or by interference with the activities of peptidoglycanspecific enzymes Among these uncertainties is one excellent example of a structure that acts as an external skeleton Borrella burgdorferl normally shaped like a flattened spiral loses its undulating shape and becomes a straight rod upon inactivation of the flaB gene which eliminates a set of periplasmic flagella Motaleb el al 2000 Evidently inter action between the flagella and peptidoglycan forces an otherwise rodshaped cell to adopt this wavy morphology Motaleb elal 2000 Although this is the bestdocu mented external mechanism for altering the gross mor phology of a cell the original shape and diameter of the wall is not dictated by the flagella Therefore the underly ing shape must be derived from some other mechanism F152 and cell shape It seems those prokaryotes we know best rely heavily on internal scaffolds to create stable heritable shapes How ever instead of behaving as rigid skeletons preliminary evidence suggests these structures direct the positioning of peptidoglycansynthesizing enzymes which in turn construct the cell s rigid external shell CarballidoLopez and Errington 2003 Kroos and Maddock 2003 We know for certain FtsZ performs this function Bacterial division is initiated at a cell s midpoint by formation of a polymeric ring of FtsZ onto which a cascade of proteins assembles to direct invagination of the envelope Bud delmeijer and Beckwith 2002 As part of this process the penicillinbinding protein PBP 3 Ftsl redirects pep tidoglycan synthesis from a diffuse incorporation through out the cylindrical part of the wall directed by PBP 2 to a localized incorporation at the developing septum Holtje 1998 Nanninga 1998 A simple though speculative explanation for this cycle isthat association with the Z ring activates PBP 3 so that it briefly outcompetes PBP 2 for substrates or other components of the synthetic appara tus In any case FtsZ polymerization constitutes the specific recurring signal that directs the location and syn thesis of septal peptidoglycan Abundant indirect evidence supports the idea that FtsZ also affects overall cell shape A long history of secondary observations indicates that aberrantly shaped E collare associated with treatments or mutations that interfere with cell division DNA replication and peptidoglycan synthe 574 K D Young sis Although the variety of causation makes it difficult to discern a common mechanism reinterpreting the results in light of what we now know suggests that altering the position or activity of FtsZ may be the unifying theme F 52 Strong evidence of a role for FtsZ comes from the serious morphological anomaliesthat arise in certain FtsZ mutants of E coli For example at the restrictive temperature an fts22ts mutant grows as branched filaments short Y forms and cells with polar protrusions and an fts226ts mutant formsY shaped cells or cells with malformed poles Bi and Lutkenhaus 1992 Addinall and Lutkenhaus 1996 A slight increase in the amount of mutant FtsZ84 protein produces branching in 2 10 of E coii cells Yu and Margolin 2000 and overproduction of another mis sense FtsZ mutant also leads to blebs and branching J Stricker pers comm Even wildtype FtsZ produces mor phological effects in some instances Overproduction of the Hhizobium meliioti FtsZ2 protein in E coii creates curved coiled and spiralshaped cells Margolin and Long 1994 overproduction of FtsZ in H meliioti produces a population in which nearly all cells have bulges or knobs are Y or T shaped or are multiply branched Latch and Margolin 1997 and constitutive FtsZ expression produces bifurcated stalks in Cauiobacter crescentus Quardokus et ai 1996 Taken together the results imply that a common FtsZdependent mechanism generates shape abnormalities and branching across genera Septation proteins Once FtsZ initiates the process the rest of the septal ring assembles by successive incorporation of additional pro teins Buddelmeijer and Beckwith 2002 Mutation of some of these also gives rise to shape defects For exam ple a truncated ftsA gene product produces highly curved Cshaped cells some of which form long convoluted corkscrew filaments Gayda etai 1992 and cells over producing four different carboxy terminal deletion mutants of FtsA are similarly curved and coiled Yim eta 2000 Also an FtsL mutant another ring component grows as Y shaped and branched cells Guzman et ai 1992 lnter estingly Z rings are destabilized in the absence of the early septation proteins ZipA or FtsA Pichoff and Lutken haus 2002 leaving open the possibility that the shape effects of defective septal rings may be explained by abnormal activity of FtsZ Min proteins and DNA replication Accurate localization of FtsZ rings also seems important for proper cell shape Normally polymerization and place ment of FtsZ is controlled by the MinCDE proteins which inhibit Zring formation at the poles and by the positions of chromosomes which restrict Zring formation to nucle oid free areas Yu and Margolin 1999 Lutkenhaus 2002 Therefore interfering with either of these two regulatory mechanisms may alter cell shape indirectly via mislocal ization of complete or partial Z rings Numerous observations argue that these two systems play some role in cell shape For example in a minB minCDE deletion mutant 2 8 of the cells are branched knobbed kinked or filamented Akerlund eta 1993 Of special note is that the earliest reports of mor phological alterations in E coii are associated with treat ments that inhibited DNA replication see References in Akerlund etai 1993 Thymine starvation of a thy strain produced cellular monsters including short filaments with knobs bulges kinks and branches Zaritsky 1977 and mitomycin Ctreated E coii C cells exhibited knobs bifurcations and internal bulges Suit etai 1967 When Akerlund etal 1993 altered chromosomal dynamics by driving DNA replication from an intH1 origin swollen and irregular cells appeared with branches knobs and kinked filaments Similarly inhibiting DNA synthesis in Agrobac terium tumefaciens caused every cell to elongate and branch Fujiwara and Fukui 1974 and 50 of mitomycin Ctreated H meliioti cells were branched Latch and Mar golin 1997 Akerlund etal 1993 speculated that such misshapen cells might arise because aberrantly located nucleoidsinterfere with the formation of a functional and properly oriented septation site Akerlund etai 1993 Consistent with this interpretation thyminestarved mutants display asymmetric invaginations in nucleoidfree regions Woldringh etai 1994 and Z rings form only between nucleoids in a min mutant and in a parC mutant defective in chromosomal segregation Yu and Margolin 1999 An easy way to reconcile these diverse observa tions is to imagine that Min mutants or imprecise chromo some segregation create these morphological oddities via the same secondary effect of aberrant Zring localization Peptidogiycan synthesis Whatever mechanism directs cell shape does so by syn thesizing the rigid peptidoglycan exoskeleton Therefore anything altering peptidoglycan formation could conceiv ably affect cell shape It is a surprise then that few muta tions of the high molecular weight penicillinbinding proteins HMW PBPs yield morphologically abnormal cells One PBP 3 mutant produces pointed poles in E coii Taschner et ai 1988 and deletion of one or more HMW PBPs from B subtiiis produces abnormal cell shapes Popham and Setlow 1996 Pedersen eta 1999 McPherson and Popham 2003 Interestingly in the case of a B subtiiis mutant lacking PBP 1 FtsZ rings are 2003 Blackwell Publishing Ltd Molecular Microbiology 49 571 580 tooattzed tmptopetty ot ate sttuotutatty abettaht th oyet 20 ot the oetts Pedevsen etat t 99 so that once agam mtstooattzattoh ot z tthgs ts oottetatea wtth shape abhotmattttes PepIdoglycan mod ea hon the Worst shapes At thts potht we need to cohstdet a ctass ot mutants that et have a demons d cormecttoh to FtsZ o o 9 dtsptay a ta a shapes that mtttot the ohataotettstto totms ot mahy dMevr eht pvokavyotes th 2 Some oetts ate shott tods ooooobaotttt some ate smatt sphetes otassto oooot me ate mutttpty btahohed m oetes some ate htghty tttegutat ot pvoduce btmvcated Baozehashape 575 tettat shapes attses by yattattohs oh a common motpho togtoat pathwa Because the LMW PBPs ate hot essehttat tot E 001 u tt byte oyt th tmthat oatahthe ttom the ends ot pepttde stdeohaths DDsc tboxypeptt dase BP 5 B 5 a a DaoD ot by t aythg otossa hnked stde ths p ttdases a a 7 Howey tgge The etove these ptoteths mus1 m uence e mech t ms t e a Xy epth saooutus The tdea that these PBPs atteot motphotogy yta The inen patches idea The totegoth oohstdetattohs make tt easy to ytsuattze 9 d 6m 3 togethet these dtspatate themes and obsetyattohs The Flg 2 Dtyetstty ot shapes th Esohehohta colt pEmc hnrbmdmg pvor teth mutants Photos at seteetea oetts wete oompttea hom seyetat mutttpte Paps the tmponahtoommoh eho t tbethgthat atHackPBPsmenomeataLtQQQ Netson andVoung ZOO Nelson dVotm 2001Anyones11ampvoduces ahas onmeht DES andnooneshapetspEcuhaHoapamcu av ttt tath tt The ptemtse ptesehtea hete tsthts oetts estabttsh thett gtoss m y by z dep ha ht meohahtsm syht tzes sttategtoatty ptao a patohes ot swaths ot thett stabte p tt gtycan ah they mathtath thett shape scattotdsde ht meohaht that at ots the Syn39 thests ot metabottoatty extbte pepttaogtyoah Inez pepztdogryoah PG 3 baozeha poles Pepttdogtyoah syhthests th E 501 and th B subMs ptoa ceeds e tehgth ot e oh t a etat t t tatthgh eta team a Pedvo eta t t9 7 Ho eVeL pepttdogtyoah at the pote t stabtee septatto t 0 pteted new matettat ts hot thsettea aha potat pepttdogtyoah ts etthet hot te ot a t ts tea tea a mety t t ctathesmtmah e I 389 de Pedvo e art tag No one Kn ws hat the potes thett Atthough the hemtoat oompot h tthe pot s e to be stmttat to to e the test ot the watt thett thveerdtmenstonat Stmcluve coma oh the pot ate mote thdertypE E cotthah those vasEMEd hete ThecoHage 2003 BtackweH Pubttshthg Ltd MoewarIItcmbtobgy 49 57mm 576 K D Young enzymes an idea supported by the fact that outer mem brane over the poles is as stable as is peptidoglycan de Pedro etal 1997 de Pedro etal 1997 dramatically advanced the mea surement and understanding of this inert material by developing a sensitive method for tracking stable pepti doglycan In this technique preexisting peptidoglycan is labelled with Dcysteine and is detected in isolated sacculi with antibiotin antibody In a series of pulsechase exper iments de Pedro etal 1997 expanded previous obser vations by establishing that the poles of E coli are metabolically inert and segregate conservatively for at least five generations the boundary between the pole and cylindrical wall being remarkably sharp de Pedro etal 1997 Strikingly rings of inert peptidoglycan were synthe sized at potential septation sites in filaments of several temperature sensitive division mutants lnert hoops appeared in strains carrying mutations affecting FtsA FtsQ and Ftsl PBP 3 but not in an FtsZts mutant and not in cells perturbed for DNA replication de Pedro etal 1997 Thus a ring of iPG may represent the earliest visible stage of septal differentiation and FtsZ either alone or via unknown proteins may initiate synthesis of this uniquely situated peptidoglycan Rothfield 2003 iPG and morphological defects Recently an important relationship was established between this inert peptidoglycan and morphological abnor malities in aberrantly shaped E colide Pedro et al 2003 As mentioned previously cells lacking multiple LMW PBPs are extremely abnormal When the location of iPG was measured in sacculi of one such mutant distinct patches of inert material appeared at positions where malformed cells exhibited kinks bends or branches Even small bulges and irregularities were associated with longlived seg ments of iPG de Pedro etal 2003 Two observations are especially informative First iPG was always associated with the original poles and with ectopic poles of branched cells Second in several cases iPG appeared to survive as partial rings incomplete arcs or patchy elements in the cylindrical portion of the wall More over these relatively largescale aggregates seemed to persist longer in mutantsthan in the parent strain de Pedro et al 2003 A similar phenomenon may occur in B subtilis mutants lacking PBP 1 Pedersen etal 1999 McPherson and Popham 2003 These mutants are thin and bent and about onethird have abnormal septa or unusually thick circumscribed deposits of peptidoglycan along the interior surface of their walls The nature and stability of these deposits is unknown but they are often associated wit 39 nitnrntinnn 39e t of interrupted invagination implying they were laid down by an aborted FtsZdependent process The strong correlation of iPG with morphological abnor malities suggests that these inert patches or swaths con stitute imperfections in the wall which distort the local geometry of the sacculus If the iPG fragments are inflex ible they will dictate the shape adopted by new pepti doglycan polymerized in their vicinity Thus synthesis of iPG at inappropriate positions would explain the shape anomalies in PBP mutants and may explain the morpho logical deficiencies of other mutants as well A shape idea The framework inert peptidoglycan From the above observations it is but a small step to propose that the regulated synthesis and localization of inert peptidoglycan determinesthe gross shape of normal cells By definition these inert fragments would be rela tively impervious to normal recycling mechanisms and would behave as sturdy longlived braces or supports Intracellular turgor pressure would govern the shape of recyclable peptidoglycan rPG located between these rigid segments but overall cell shape would be dictated by the geometrical arrangement of inert swaths or patches Establishment FtsZ localizes iPG synthesis As discussed earlier mutations and manipulations that alter the activity or localization of the E coil Z ring often give rise to unusually shaped cells The data are most easily harmonized by presuming that FtsZ plays an unspecified role in morphology In view of the tight asso ciation between inert peptidoglycan and the morphological discontinuities in misshapen cells the simplest interpreta tion is that FtsZ participates in synthesis and localization of iPG This is probably true during normal cell division because the earliest observable step in septal develop ment seems to be the FtsZdependent synthesis of a hoop of iPG de Pedro etal 1997 It is reasonable to suppose that if conditions allow FtsZ to polymerize elsewhere then partial Z rings or incomplete arcs could initiate iPG syn thesis at atypical positions Should they persist these bits of misplaced or extended iPG may develop into the ectopic poles and inflection points of aberrantly shaped cells Mor phological irregularities would arise from a combination of the frequency of FtsZ polymerization at uncharacteristic sites the lifetimes of such polymers and the ability of these structures to activate iPG synthesis It must be noted that this emphasis on the role of FtsZ in cellular morphology seems at odds with the conclu sions drawn by Gullbrand etal 1999 These authors observed branching at the restrictive temperature in E coli mutants expressing the temperaturesensitive FtsZ84 protein and saw no consistent association of Z rings with 2003 Blackwell Publishing Ltd Molecular Microbiology 49 571 580 branch points suggesting that FtsZ plays at best a minor role in this process Gullbrand etal 1999 However their results and the ideas expressed here can be recon ciled if nucleation of a branch point and elaboration of a branch are separate events In this view transient poly merization of Fts284 would initiate synthesis of iPG at inappropriate sites after which the responsible Z ring or incomplete Z arc would disappear to reassemble else where The extra patch of iPG may then behave as an ectopic pole and continued peptidoglycan synthesis would extend it creating a branch Thus once formed misplaced iPG would give rise to branches in the absence of active Fts284 Just as Gullbrand et al 1999 conclude aberrant septation per se would not be responsible for branching However incomplete or inaccurate Zring activity would still be the decisive factor that generates iPG which would constitute the previous cell wall abnor malities that the authors surmise lead to branching Gull brand etal 1999 Maintenance scaffolding localizes rPG Although iPG may determine overall cell shape by provid ing a framework other internal or external scaffolds must still guide the shape of newly synthesized peptidoglycan connecting these molecular girders Synthesis of a uni Bacterial shape 577 form cylindrical wall certainly depends on the activities of FtsZ MreB Mbl and other proteins The dimensions of a preexisting wall may influence the exact geometries of these internal scaffolds which may in turn position the peptidoglycan synthetic apparatus so as to assemble a sacculus of defined shape Agents external to the wall may act similarly or as in the case of the flagella of B burgdorferi may impose an additional tertiary structure Motaleb etal 2000 Controlling iPG synthesis How might a cell regulate the judicious arrangement of iPG to create a specific shape Potential mechanisms could i affect the supply or localization of precursors ii dictate the location rate of formation quantity or chemical nature of glycan chains and crosslinks or iii modify the location rate or extent of peptidoglycan degradation Fig 3 As described above FtsZ would drive the second mechanism The first and third would be mediated by the LMW PBPs Substrate availability The DDcarboxypeptidase activity of PBP 5 removes the Precursors Glycan composition Product Cell PBP 5 NAGTAM Recyclable Uniform gt Tri Jgt Peptidoglycan D Shape Tetra NAGJTAM Penta peptides Penta e tides PBP 4 pp PBP5 NAG NAM I FtSZ Inert Aberrant Pentapept39des Peptidoglycan Shape l 1 t l 1 Supply Localization Degradation Growth amp Synthesis Fig 3 Hypothetical steps in the synthesis and degradation of inert peptidoglycan in E coli Muropeptides Precursors are synthesized in the cytoplasm and delivered to synthetic enzymes in the periplasm where they are polymerized and crosslinked Glycan composition Different enzymes create either recyclable or inert peptidoglycan Product It is not known whether PBP 5 uses monomer precursors or polymeric peptidoglycan or both as substrates so both alternatives are illustrated Supply Although PBP 5 is the most active PBP at the steps indicated other DDcarboxypeptidases PBP 4 PBP 6 or DacD may also contribute The participation of FtsZ Localization and Synthesis is hypothetical and its exact function is unknown The removal of inert peptidoglycan Degradation is hypothetical and may be accomplished by PBP 4 unpublished or by other hydrolases endopeptidases amidases or lytic transglycosylases Such modification of inert peptidoglycan could create a recyclable form top product or the modified components could be degraded and recycled to the cytoplasm not shown These degradative enzymes might also act earlier to remove a substrate required by the FtsZdependent step NAG Nacetylglucosamine NAM Nacetylmuramic acid PBP penicillinbinding protein Pentapeptide Lalay DgluDapDalaDala Dap diaminopimelic acid Open arrows indicate activity of peptidoglycan synthetic enzymes closed arrows indicate hypothetical activity of the indicated proteins 2003 Blackwell Publishing Ltd Molecular Microbiology 49 571 580 578 K D Young terminal Dalanine from a percentage of pentapeptide sidechains creating a tetrapeptide that can act only as a recipient during subsequent crosslinking Fig 3 Supply Holtje 1998 Elimination of PBP 5 increases the concen tration of pentapeptides and the pool of available donor sidechains which must be critical because PBP 5 is the most influential low molecular weight PBP in terms of generating misshapen cells in multiple mutants Nelson and Young 2000 Nelson and Young 2001 Remember ing that deformities in such mutants correlate with an increase in iPG de Pedro el al 2003 these phenomena are incorporated into the hypothetical scheme of Fig 3 by proposing that PBP 5 regulatesthe level of a pentapeptide substrate required for iPG synthesis Perhaps a similar phenomenon accounts for morphological changes observed in a B sublilis mutant impaired in isoprenoid biosynthesis which is required for delivery of precursors for peptidoglycan and teichoic acid synthesis Campbell and Brown 2002 Removing excess iPG We may expect that some superfluous inert peptidoglycan is synthesized no matter what controls its production An E colimutant with a high percentage of grossly deformed cells requires deletion of genes encoding at least three PBPs one of which must be PBP 5 Nelson and Young 2001 For example a strain lacking PBPs 4 5 and 7 produces extraordinarily irregular cells PBPs 4 and 7 are endopeptidases that cleave preexisting crosslinks between glycan chains of peptidoglycan Therefore a sig nificant morphological effect occurs when at least two different functions are removed from the cell If in the absence of PBP 5 PBPs 4 and 7 cleave and remove inadvertently synthesized iPG the effect of losing PBP 5 may be masked Fig3 Eliminating this degradative path way in a triple mutant may allow iPG fragments to persist long enough to be incorporated into the wall creating shape deformities Although the above posits that PBPs 4 and 7 remove iPG any of a number of peptidoglycan hydrolases 39 midases might do the same Holtje 1998 Of course the scenario leaves many unanswered questions Why don t the hydrolases remove all inert peptidoglycan including that synthesized at the site of septation Is their capacity for iPG removal limited Might they act only on iPG below a certain size threshold ls iPG distinguishedfrom rPG by acertaintype of crosslinking or is iPG inert for some other reason but still susceptible to hydrolysis Is the iPG converted to rPG or is it degraded to monomers and recycled via the cyto plasm Fortunately these and other questions growing out of this proposed scheme can be addressed experimentally Concluding remarks A decade ago Akerlund elal 1993 suggested that the underlying control circuitry and the cell division compo nents of rodshaped and branching bacteria may be more similar than expected In fact many prokaryotes may share large portions of a common morphogenetic appa ratus suitably adapted to sustain a form selected from the universe of possible shapes To create any desirable shape a cell need only possess an iPG synthesizing apparatus and be able to control its movement and the activity of the enzymes within it Of course the mecha nisms restricting enzyme activity to specified areas of the cell await a complete description but the recent conflu ence of results should begin to answer longheld ques tions about bacterial shape Acknowledgements lthankthe members of my laboratory past and present who have worked so diligently to ferret out the functions of under appreciated PBPs I also thank Margaret Larson and Rebecca Thurn for library work Kim Young and John Lee for graphics and Ann Flowerfor commenting on the manuscript I apologize to those authors I could cite only indirectly via previous reviewsThe quotation from John Ruskin is from The Columbia World of Quotations Andrews et al 1996 This work was supported by grant GM61069 from the National Institutes of Health and by grant MOB9982157 from the National Science Foundation Note added in proof While this review was in press Shih et al reported that the MinODE proteins also form distinct spiral structures within the eytoplasm of E coil Shih YL Le T and Rothfield L 2003 Prac NatlAcad Sci USA online publication 101073 pnas1232225100 The Min spirals differ from those formed by Mch and arc dynamic oscillating from pole to pole within the cell The same considerations apply to these spirals as discussed in the text regarding the use of other proteins as internal scaffolds References Abhayawardhane Y and Stewart GO 1995 Bacillus subtilis possesses a second determinant with extensive sequence similarity to the Escherichia cali mreB morpho gene J Bacterial 177 765 773 Addinall SG and Lutkenhaus J 1996 FtsZ spirals and arcs determine the shape of the invaginating septa in some mutants of Escherichia cali Mal Microbial 22 231 237 Akerlund T Nordstrom K and Bernander R 1993 Branched Escherichia cali cells Mal Microbial 1 0 849 858 Andrews FL Biggs M and Seidel M eds 1996 The Columbia World of Quotations New York Columbia Uni versity Press 2003 Blackwell Publishing Ltd Molecular Microbiology 49 571 580 Belaaouaj A Kim KS and Shapiro SD 2000 Degrada tion of outer membrane protein A in Escherichia coli killing by neutrophil elastase Science 289 1185 1188 Bi E and Lutkenhaus J 1992 Isolation and characteriza tion of ftsZalleles that affect septal morphology J Bacteriol 174 5414 5423 Buddelmeijer N and Beckwith J 2002 Assembly of cell division proteins at the E coli cell center Curr Opin Micro biol 5 553 557 Campbell T and Brown E 2002 Characterization of the depletion of 2CmethylDerythritol24cyclodiphosphate synthase in Escherichia coli and Bacillus subtilis J Bacte riol184 5609 5618 CarballidoLopez FL and Errington J 2003 The bacterial cytoskeleton in vivo dynamics of the actinlike protein Mbl of Bacillus subtilis Dev Cell 4 19 28 ClarkeSturman AJ Archibald AFL Hancock lC Har wood CFL Merad T and Hobot JA 1989 Cell wal assembly in Bacillus subtilis partial conservation of polar wall material and the effect of growth conditions on the pattern of incorporation of new material at the polar caps J Gen Microbiol 135 657 665 Cooper S 1991 Bacterial Growth and Division Biochem istry and Regulation of Prokaryotic and Eukaryotic Division Cycles San Diego Academic Press Inc Denome SA Elf PK Henderson TA Nelson DE and Young KD 1999 Escherichia coli mutants lacking all possible combinations of eight penicillin binding proteins viability characteristics and implications for peptidoglycan synthesis J Bacteriol181 3981 3993 van den Ent F Amos LA and Lowe J 2001 Prokaryotic origin of the actin cytoskeleton Nature 413 39 44 Fujiwara T and Fukui S 1974 Unidirectional growth and branch formation of a morphological mutant Agrobacte rium tumetaciens J Bacteriol 120 583 589 Gayda FLC Henk MC and Leong D 1992 Cshaped cells caused by expression of an ttsA mutation in Escher ichia coli J Bacteria174 5362 5370 Gullbrand B Akerlund T and Nordstrom K 1999 Onthe origin of branches in Escherichia coli J Bacteriol 181 6607 6614 Guzman LM Barondess JJ and Beckwith J 1992 FtsL an essential cytoplasmic membrane protein involved in cell division in Escherichia coli J Bacteriol 174 7717 7728 Harold FM 1990 To shape a cell an inquiry into the causes of morphogenesis of microorganisms Microb Rev 54 381 431 Hegermann J Herrmann FL and Mayer F 2002 Cytosk in k Bacterial shape 579 Holtje JV 1998 Growth of the stressbearing and shape maintaining murein sacculus of Escherichia coli Microbiol Mol Biol Rev 62 181 203 Jones LJ CarballidoLopez FL and Errington J 2001 Control of cell shape in bacteria helical actinlike filaments in Bacillus subtilis Cell 104 913 922 Koch AL 1988 Biophysics of bacterial wall viewed as a stressbearing fabric Microb Rev 52 337 353 Koch A L 2000 Simulation ofthe conformation of the murein fabric the oligoglycan pentamuropeptide and cross linked nonamuropeptide Arch Microbiol174 429 439 Kroos L and Maddock JR 2003 Prokaiyotic develop ment emerging insights JBacteriol185 1128 1146 Latch JN and Margolin W 1997 Generation of buds swellings and branches instead of filaments after blocking the cell cycle of Rhizobium meliloti J Bacteria179 2373 2381 Lu C Reedy M and Erickson HP 2000 Straight and curved conformations of FtsZ are regulated by GTP hydrol ysis J Bacteriol182 164 170 Lutkenhaus J 2002 Dynamic proteins in bacteria Curr Opin Microbiol 5 548 552 Lutkenhaus J and Addinall SG 1997 Bacterial cell divi sion and the Z ring Annu Rev Biochem 66 93 16 Margolin W and Long SR 1994 Rhizobium meliloti con tains a novel second homolog of the cell division gene Ftsz J Bacteriol 176 2033 2043 McPherson DC and Popham DL 2003 Peptidoglycan synthesis in the absence of class A penicillinbinding pro teins in Bacillus subtilis J Bacteriol185 1423 1431 Mendelson NH 1982 Bacterial growth and division genes structures forces and clocks Microb Rev 46 341 375 Merad T Archibald AFL Hancock lC Harwood CFL and Hobot JA 1989 Cell wall assembly in Bacillus sub tilis visualization of old and new wall material by electron microscopic examination of samples stained selectively for teichoic acid and teichuronic acid J Gen Microbiol 135 645 655 Motaleb MA Corum L Bono JL Elias AF Rosa P Samuels DS and Charon NW 2000 Borrelia burgdor teri periplasmic flagella have both skeletal and motility functions Proc NatlAcad Sci USA 97 10899 10904 Nanninga N 1998 Morphogenesis of Escherichia coli Microbiol Mol Biol Rev 62 110 129 Nelson DE and Young KD 2000 Penicillin binding pro tein 5 affects cell diameter contour and morphology of Escherichia coli J Bacteriol182 1714 1 721 Nelson DE and Young KD 2001 Contributions of PBP 5 and DDcarboxypeptidase penicillin binding proteins to Naturwissenschatten 89 453 458 Henning U 1975 Determination of cell shape in bacteria Annu Rev Microbiol 29 45 60 Henning U and Schwarz U 1973 Determinants of cell shape In Bacterial Membranes and Walls Leive L ed New York Marcel Dekker Inc pp 413 438 Hiemstra H Nanninga N Woldringh CL lnouye M and Witholt B 1987 Distribution of newly synthesized lipopro tein over the outer membrane and the peptidoglycan sac culus of an Escherichia coli laclpp strain J Bacteriol 169 5434 5444 2003 Blackwell Publishing Ltd Molecular Microbiology 49 571 580 shape in Escherichia coli J Bacteriol 183 3055 3064 Ohara M Wu HC Sankaran K and Rick PD 1999 Identification and characterization of a new lipoprotein Nlpl in Escherichia coli K12 JBacteriol181 4318 4325 Pas E Einav M Woldringh CL and Zaritsky A 2001 Perpendicular planes of FtsZ arcs in spheroidal Escheri chia coli cells Biochimie 83 121 124 Pedersen LB Angert EFL and Setlow P 1999 Septal localization of penicillinbinding protein 1 in Bacillus subti lis J Bacteriol181 3201 321 1 de Pedro MA Quintela JC Holtje JV and Schwarz H 580 K D Young 1997 Murein segregation in Escherichia cali J Bacterial 179 2823 2834 de Pedro MA Donachie WD Holtje JV and 8chwarz H 2001 Constitutive septal murein synthesis in Escheri chia cali with impaired activity of the morphogenetic pro teins RodA and penicillinbinding protein 2 JBacterial183 4115 4126 de Pedro MA Young KD Holtje JV and 8chwarz H 2003 Branching of Escherichia cali cells arises from mul tiple sites of inert peptidoglycan J Bacterial 185 1147 1152 Pichoff 8 and Lutkenhaus J 2002 Unique and overlap ping roles for ZipA and FtsA in septal ring assembly in Escherichia cali EMBO J 21 685 693 Popham DL and 8etlow P 1996 Phenotypes of Bacillus subtilis mutants lacking multiple class A highmolecular weight penicillinbinding proteins J Bacterial 178 2079 2085 Quardokus E Din N and Brun YV 1996 Cell cycle regulation and cell typespecific localization of the FtsZ division initiation protein in Caulabacter Prac Natl Acad Sci USA 93 6314 6319 Rothfield L 2003 New insights into the developmental his tory of the bacterial cell division site J Bacterial 185 1125 1127 8chwarz U and Leutgeb W 1971 Morphogenetic aspects of murein structure and biosynthesis J Bacterial 1 06 588 595 80nntag l 8chwarz H Hirota Y and Henning U 1978 Cell envelope and shape of Escherichia cali multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins JBacterial136 280 285 Suit JC Barbee T and Jetton 8 1967 Morphological changes in Escherichia cali strain C produced by treat ments affecting deoxyribonucleic acid synthesis J Gen Micrabial49 165 173 Sun 0 and Margolin W 2001 Influence of the nucleoid on placement of FtsZ and MinE rings in Escherichia cali J Bacterial 183 1413 1422 Taschner PE Ypenburg N 8pratt BG and Woldringh CL 1988 An amino acid substitution in penicillinbinding protein 3 creates pointed polar caps in Escherichia cali J Bacterial 170 4828 4837 Wachi M Doi M Tamaki 8 Park W Nakajimalijima 8 and Matsuhashi M 1987 Mutant isolation and molecular cloning of mre genes which determine cell shape sensi tivity to mecillinam and amount of penicillinbinding pro teins in Escherichia cali J Bacterial 169 4935 4940 Woldringh C Huls P Pas E Brakenhoff GJ and Nanninga N 1987 Topography of peptidoglycan synthe sis during elongation and polar cap formation in a cell division mutant of Escherichia cali MC4100 J Gen Micra bial 133 575 586 Woldringh CL Zaritsky A and Grover NE 1994 Nucle oid partitioning and the division plane in Escherichia cali J Bacterial 176 6030 6038 Yim L Vandenbussche G Mingorance J Rueda 8 Casanova M Ruysschaert JM and Vicente M 2000 Role of the carboxy terminus of Escherichia cali FtsA in self interaction and cell division J Bacterial 182 6366 6373 Yu XC and Margolin W 1999 FtsZ ring clusters in min and partition mutants role of both the Min system and the nucleoid in regulating FtsZ ring localization Mal Microbial 32 315 326 Yu XC and Margolin W 2000 Deletion of the min operon results in increased thermosensitivity of an ftsZB4 mutant and abnormal FtsZ ring assembly placement and disas sembly J Bacterial 182 6203 6213 Zaritsky A 1977 Branching of fastgrowing Escherichia cali 15T at low thymine concentrations FEMS Microbial Lett 2 5 69 2003 Blackwell Publishing Ltd Molecular Microbiology 49 571 580 Review Questions2 Energy and Metabolism ATP its role in metabolism generation of ATP by different mechanisms Mention the number of high energy bonds in each of the following molecules ATP PEP 13 BPG ADP AMP catabolism anabolism endergonic exergonic reactions Redox reactions Role of electron carriers NAD NADP etc Enzymes mechanism of action effect of environment inhibition regulation Overview of metabolism Glucose to pyruvate Glycolysis Pentose Phosphate pathway TCA cycle ETCOxidative Phosphorylation anaerobic phosphorylation Fermentation organic e acceptors their role How is NAD regenerated in Oxidative phosphorylation fermentation Why is it necessary to regenerate NAD Photosynthesis CalvinDark cycle Gluconeogenesis Organization of synthetic pathways DNA structure amp replication Explain the ow of genetic information in a diagram Include the exceptions if any How did Griffith s expt with S pneumoniae demonstrate the transformation of nonvirulent strain to virulent strain Be familiar with the double helical structure for DNA Know the structures of DNA RNA and proteins What polarity do the strands have What bond is it between complementary Nbases What are Chargaff s rules Can these be used for single stranded polynucleotides For different DNA forms A B and Z forms which are right handed Which are left handed What is the role of Z form if any in human beings Complementarity rules Hbond covalent bond phosphodiester linkage SugarPhosphate backbone antiparallel Name the bond that links adjacent nucleotides in the same strand in nucleic acids Peptide Betalgt4 glycosidic 5 3 phosphodiester hydrogen bond What is the most common form of DNA found in cells What are the other forms What gives Z DNA its zig zag structure Which of the following are true about the structure of DNA a Purine and pyrimidine bases are attached to the C1 of the deoxyribose sugars b The bases extend towards the middle of the cylinder formed by the two chains c The bases are stacked on top of each other in the center with the rings forming parallel planes d All of the above are true How did Messelson and Stahl prove that DNA replication is semi conservative How many replicons does E 001139 have Is the chromosomal DNA replication bidirectional in both prokaryotes and eukaryotes Both DNA replication and transcription proceed in the 5 to 3 direction Mention one important difference between DNA polymerases and RNA polymerases with respect to initiation of polymerization True False Many prokaryotes have only one origin of replication per DNA molecule while most eukaryotes have multiple origins per DNA molecule What is required in the initiation of DNA replication What is the origin of replication What characteristic elements does it contain What is the role of Ori C DnaA Dna helicase DNA Pol I and Pol III in chromosomal DNA replication in E 001139 Why do you need a free 3 OH for chain elongation E coli DNA Pol III core and holoenzyme What are the different subunits and what are their functions Which subunit accounts for polymerizing Which subunit accounts for 3 5 exonuclease activity Which subunit accounts for processivity What is proof reading How would DNA replication differ in a cell with a mutant epsilon subunit What properties do DNA polymerase I and III share Which property is unique to DNA polymerase I Why is DNA polymerase I considered distributive and polymerase III processive What is the main difference between how the leading strand is synthesized and how the lagging strand is synthesized What are Okazaki fragments How and why are they formed What is the role of beta clamp Is it a part of the core polymerase or of the holoenzyme In leading strand synthesis DNA pol III can proofread the newly formed DNA this function is performed by subunit of the enzyme It recognizes errors removes the wrong nucleotide through 7 to 7 exonuclease activity E 001139 makes an error of only 1 in 1010 bp during DNA replication What accounts for this extreme fidelity What are the functions of the following molecules in the replication of E coli genome DnaA DnaB DnaC SSB DNA topoisomerase I DNA gyrase DNA primase DNA ligase DNA pol I DNA pol III holoenzyme What are the important functions of telomerases Are they necessary in prokaryotes Would it help E coli to have telomeres Explain What is semi 7 conservative replication What is bidirectional replication How many replicons does the bacterial chromosome have dNTP s provide energy for chain elongation during replication TrueFalse What is the role of Type I and Type II topisomerases How would you distinguish between template DNA and newly synthesized DNA Gene organization in Phage PhiXl74 is rather unusual What is it that makes it so unusual Tau and gamma subunits of DNA PolIII holoenzyme are a product of the same gene dnaX This is an example of one gene 2 products How do you think these are formed Dr Walker s work on DNA replication Result of ribosomal frame shifting Draw ds DNA Use each of the two strands strand 1 and 2 as template strands Fork movement is from left to right for strand 1 Strand 1 3 5 Strand 2 5 3 Which strand is the template for the synthesis of the leading strand Which one is the template for the synthesis of the lagging strand Name the proteins enzymes required for the synthesis of the leading strand Name the proteins enzymes required for the synthesis of the lagging strand RNA structure amp transcription Structure of m r t RNAs Special features of tRNA Unusual nucleotides Hbonded ds regions What kind of effect will be observed in cells that contain a mutant sigma 70 factor the predominant sigma factor in the cell Explain your answer How many RNA polymerases do the prokaryotes have How many do the eukaryotes have What do you understand by the term Ribozymes Mention some functions of the ribozyme What is the role of sigma factor in transcription What is the advantage of having more than one sigma factor in a cell What is the role of different subunits of E coli RNA polymerase in transcription What is the difference between core polymerase and holoenzyme in case of RNA polymerase Define promoter and terminator with respect to transcription ProEukaryotic promoters 35 10 1 start site TATA box CAAT GC box operators What are transcription start site and termination sites Pribnow box is situated at the region Give position on DNA with respect to transcription start site What are the two important types of termination of transcription The promoter is the region of the DNA to which binds DNA Pol RNA Pol ribosome Select the best answer What is the difference between rho dependent and rho independent terminators Describe the difference between Prokaryotic and Eukaryotic mRNA Post transcriptional modi cations post translational modi cations Mention which step of transcription is inhibited by rifampicin streptolydigin Write down a 20 base pair long ds DNA sequence include the promoter region 10 1 Label its 5 and 3 ends on both the strands Transcribe this DNA fragment Label the 5 and 3 ends of the transcript Translation What is the role of all 3 RNAs in translation Is DNA directly involved in translation What is meant by the term coupled transcription and translation Coupled transcription and translation takes place in prokaryoteseukaryotes Think of differences between prokaryotic and eukaryotic mRNAs What are the different steps and factors involved in all three processes initiation elongation and termination of translation What do you understand by the following terms codon codon degeneracy start and stop codon sense and nonsense codons etc Why is the genetic code considered degenerate and unambiguous What is the difference between a monocistronic and a polycistronic message What is the role of leader sequence terminator sequence and trailer sequence What is role of initiator tRNA Met or fMet tRNA Why would you expect to find fMet ONLY at the amino terminal of the polypeptide and not at the Cterminal or in the middle of the polypeptide chain What is the significance of Shine Dalgarno sequence At which step does it play a role The ShineDalgamo sequence is a stretch of nucleotides present on the 5 end of prokaryotic mRNA 5 end of eukaryotic mRNA 5 end of 16s rRNA 3 end of 16s rRNA What is the role of amino acyl tRNA What is the role of aminoacyl tRNA synthetase Why is the proofreading activity of the amino acyl tRNA synthetases significant What catalyzes the transpeptidation in translation There are 61 codons that specify amino acids These are called codons The three codons UAG UGA and UAA which do not specify amino acids are called codons and cause termination of Is the leader sequence of the mRNA translated What is its function Be familiar with the structure of ribosome What is the role of 16 S rRNA in translation The ribosome has three sites for binding tRNA molecules The initiator tRNA binds at the site the subsequent incoming tRNAs are positioned in the site RNA is transcribed in the direction Proteins are synthesized from the end to end If the mRNA first sits on the small subunit of the ribosome why is the larger subunit needed Indicate all the energy requiring process in this process What are the roles of EF Tu and EFTs Which one of these directly participates in the step of elongation during the process of translation What is the role of peptidyl transferase in the step of elongation during the process of translation What are the roles of the release factors in translation Are sigma factors needed for transcription or translation or both Mention 2 inhibitors of prokaryotic protein synthesis Erythromycin and tetracycline function by inhibiting protein synthesis In that case are these antibiotics also toxic to us humans when we take them Explain your answer Mention ANY ONE important difference between prokaryotic and eukaryotic RNAs The major function of the 23SrRNA in the larger subunit of the ribosome is Consider a hypothetical situation in which the cell has N formyl lysine Can this modified amino acid be incorporated in growing polypeptide chain If it can will it still produce a full length polypeptide or will it be a truncated product What will be the situation in the case of a Nformyl alanine Explain your answer Consider all other conditions in the cell normal Clue The question does not deal with the structural or functional implications of this polypeptide Know some inhibitors of DNA replication transcription and translation Where do replication transcription and translation take place in prokaryoteseukaryotes What are main differences similarities between prokaryotic and eukaryotic ribosomes Be familiar with the structure of initiator tRNA Why does initiator tRNA function only in initiation instead of elongation What is the difference between monocistronic and polycistronic mRNA Sketch a figure to show the difference between these messages being translated by several ribosomes at the same time Define polysomes polyribosomes What is the function of molecular chaperones What are molecular chaperones Are they present in both eukaryotes and prokaryotes Why are these referred to as heat shock proteins Why are they found to be up regulated during heating or other stressful conditions Chaperones DnaK DnaJ bind to nascent polypeptides What is their function here What is the role of sigma 32 in heat shock What is protein splicing In protein splicing inteins are regions of the polypeptide that are and eXteins are the regions that are Given below is a 20 bp long ds DNA molecule ATCGCATGGCTACTGCATAG TAGCGTACCGATGACGTATC Label the 5 and 3 ends Transcribe this DNA sequence to give the sequence of the mRNA Indicate the template strand topbottom you used to obtain this RNA Label the5 end of the mRNA Translate this mRNA to give at least three amino acids in tandem What are the different steps in a DNA replication b Transcription c Translation Label the figures for all processes showing all the proteins enzymes involved in these Gene regulation What are the different levels at which genes can be regulated How is the regulation at enzyme activity different from that at transcription translation level How is phosphofructokinase regulated Mention 2 chemicals that can regulate the activity of PFK How are the genes regulated at the mRNA level Define operon Describe induction and repression Negative Positive control Induction repression LacTrp operons Maltose regulon The synthesis of mRNA is quot J by These 39 39 are the products of regulatory genes The repressor binds to a specific site on DNA called Give two examples of genesoperons that are under negative regulation Repressors active inactive DiauXic growth phase in presence of both glucose and lactose What is the role of repressor and operator in gene regulation What is the role of cAMP CRP CAP adenyl cyclase in regulation the lac operon What are constitutive mutants What would be 2 ways to express lac trp operon constitutively How would a mutant trpR behave under different levels of trp concentration in the environment Attenuation in biosynthetic pathways Its importance esp wrt Trp operon What is the role of trp tRNA trp tRNA synthetase in regulationof trp operon by attenuation Attenuation takes place in prokaryoticeukaryotic cells Circle the correct answer What is the role of DNA binding proteins in gene regulation Why do most prokaryotic regulatory proteins have a helix turn helix motif How does this folding pattern help in recognizing the regulatory site Global regulation by different sigma factors Quorum sensing as an example of global gene regulation in bioluminescence of Vibriofzscheri biofilms formed by P aeruginosa refer Brock What is the role of sigma factors in gene regulation How are the levels of sigma 32 regulated in E 001139 How is sporulation regulated in B subtilis How is the synthesis of molecular chaperones regulated Define antisense RNA What is its role in regulating gene expression Chemotaxis Mention the roles of all the different proteins involved in the process of chemotaxis How would phosphorylation of Che A be affected by the presence of an attractant Control of cell cycle E coli cells growing with a generation time of 60 minutes or longer have a discontinuous synthesis of chromosomal DNA whereas those growing faster have continuous DNA synthesis Truefalse What is the role of FtsZ in cell division How do cell mass and cell length regulate DNA replication and cell division In positive control when cAMP is absent or present at low level the CAP protein remains activeinactive and does not bind to the Thus RNA polymerase does not bind to the and transcribe the genes in that operon True false In inducible systems the repressor protein is active until bound to the inducer where as in repressible systems the repressor is inactive until bound to the corepressor Define attenuation and attenuator Where is the leader region located What does it contain What is the role of leader peptide in the control of transcription In the leader region of trp operon what is the consequence if a mRNA segment 1 and 2 pair to form a hairpin and segment 3 and generate a second hairpin loop b Segment 1 is prevented from base pairing with segment 2 segment 2 associates with segment 3 and segment 4 remains single stranded Describe the control of tryptophan operon function by attenuation in the following cases a No translation of mRNA b Tryptophan available to form trp tRNA c Tryptophan absentpresent in low amounts What is catabolite repression Why is a biphasic growth curve observed when bacteria are grown in the presence of glucose and lactose Is the control of the lac operon by the catabolite activator protein CAP an example of positive or negative regulation Distinguish between negative and positive regulation Draw the diauxic growth curve of e coli grown in the presence of both glucose and lactose Why does E coli use lactose only after it has used up the glucose present in the medium How does the attenuator help in regulating the expression of the Trp operon in low levels of tryptophan Use of different types of sigma factors by the cell is a means of global regulation of genes Explain taking any example discussed in class as a model Mention the role of sigma 32 in the formation of heat shock proteins to protect the cell from thermal destruction Review Questionsl General concepts historv What was the technique that Carl Woese used to identify another domain to classify mo in How did Pasteur help resolve the debate on spontaneous generation What is the difference between Pasteurization and Tyndallization What kinds of organisms survive pasteurization How is pasteurization different from sterilization What was the contribution of Carl Woese to the classi cation of organisms What domains did Woese divide microorganisms into How did his research revolutionize the phylogenetic tree of microorganisms Why did Carl Woese choose rRNA as a molecule that could be studied for phylogeny What other molecules could be used instead What surprising results did his study bring up Know the names and characteristics of some organisms 2 large 2 typical and 2 unique in some way other than the large size from the table in BIG BACTERIA What is the importance of Koch s postulates What are the molecular Koch s postulates How do they reinforce the original criteria that Koch put forward in determining the causal relationship between microorganisms and disease Why was the development of solid media a boon to microbiology Why did Agar quickly overtake gelatin as the most favored solidifying agent Microscopy and staining Compare and contrast between light microscope and electron microscope both TEM and SEM Know the resolutions limits and magni cations achieved by each Since electron microscopes have higher magni cation as compared to light microscopes can you use it to observe a moving paramecium What other kinds of microscopy can you use to observe such a specimen What are the different kinds of electron microscopes Which kind would you use to study a internal structure of the mitochondria b morphology of a bacterial cell Why is Gram staining considered a differential staining technique Know the procedure of Gram staining What is the current understanding about why Gram negative organisms do not retain the primary stain Why is it dif cult to stain Mycobacterium tuberculosis using Gram staining technique Acidfast staining is used speci cally on bacterial strains that contain in their cell wall Cell Structure and Function What are the three basic shapes of bacterial cells Do all cells have rRNA What is the purpose of rRNA What is the typical size of a prokaryotic cell Compared to the typical size how many times is Epulopiscium shelsom39 bigger in volume What is the theoretical minimum genome size of a freeliving organism What are Nanobacteria Would you expect Nanobacteria to be freeliving cells Explain How are big bacteria like Thiomargarita namebiensis and Epilopz39scz39um shelsom39 able to survive as prokaryotes in spite of their huge sizes Know the functions of Prokaryotic and Eukaryotic structures In which way are the prokaryotic ribosomes similar to the eukaryotic ribosomes and in which way are these different What is nucleoid What are plasmids What is the major chemical component that differs in the plasma membrane of prokaryotes and eukaryotes What is its function in the membrane What function does cholesterol have in the cell membranes of eukaryotes What do bacteria have instead of cholesterol in their membranes What makes the plasma membrane amphipathic What makes the plasma membrane selectively permeable What is the MAIN difference between a eubacterial and an archaebacterial plasma membrane Give two distinguishing characteristics of the Archael membranes What are the similarities and differences between passive diffusion facilitated diffusion active transport and group translocation What is passive transport What kinds of molecules enter the cell through this kind of transport Do you think that amino acids are passively transported into the cell What is facilitated diffusion How is it different from active transport Why do microorganisms normally take up nutrients using transport proteins or permeases What advantage does a microorganism gain by employing active transport rather than facilitated diffusion How are molecules transported by the ABC transport mechanism How does the PTS transport system function In what way is this system different from the ABC system Which one of the transport systems is defective in patients having cystic fibrosis Compare in a table the similarities and differences between passive diffusion facilitated diffusion ABC transport and group translocation Prokamotic cell wall Sketch a Gram ve and a Gram ve cell wall Highlight the characteristic features of each How do the Mycoplasmas differ from the G ve and G ve cells What is unique about the cell wall of Mycobacterium What is periplasmic space What is the nature of the proteins that are present in the periplasmic space What are the major components of the cell walls of Plants Fungi Eubacteria Archaebacteria What is the advantage in having D amino acids in the cell wall What structure accounts for the negative charge and for toxicity in G ve cells Know in detail the structure of Gram positive and Gram negative cell walls What is the difference in the type of crosslinking found in the peptidoglycan of most Gram positive and Gram negative bacteria What is characteristic of the third amino acid in the peptidoglycan subunit What other amino acid can take the place of DAP or lysine Why What are the components of LPS What is the function of 0 antigen What makes LPS an endotoxin How is the outer membrane different from the plasma membrane Why are Archaebacteria resistant to the action of lysozyme What is the major difference between the cell wall of Archae and that of Eubacteria The polymerization of the new subunit of the peptidoglycan to the existing cell wall occurs outside the plasma membrane Where does the energy for the transpeptidation step come from What is the function of bactoprenol What are autolysins Why are they important What is the role of autolysins in the synthesis of bacterial cell walls Mention the different patterns of cell wall formation What is the role of penicillin binding proteins in cell wall synthesis Describe the role of Uridine diphosphate and bactoprenol in peptidoglycan synthesis Be familiar with the steps in PG synthesis Mention the step s at which he following inhibitors act Cycloserine penicillin vancomycin and bacitracin Include in our answer whether these steps take place in cytoplasm or outside it ie whether the substance needs to enter the cytoplasm to function or not Penicillin inhibits transglycosylation and transpeptidation True False Mention one inhibitor that inhibits both the processes What are the similarities and differences between penicillin and lysozyme in terms of their action on the prokaryotic cell Think of their action on the cell wall and whether these will work on growingstationary cells What is the site of action of a lysozyme b penicillin What would happen to protoplasts in an isotonic hypotonic and hypertonic environment If the cell wall is ruptured what condition must be provided in order to keep the cell from lysing Capsule Flagella Endospore What is a capsule What is it usually made up of Name important advantages provided to the bacterial cell by the capsule In what ways does the capsule help the cell What is the composition macromolecule of capsule Why does the smooth form of S r 39 cause 1 39 whereas the rough form does not I What are the different patterns of agella distribution What is the function of agella in bacteria How is the agella synthesized How does agella grow Be familiar with the agellar ultra structure Know the composition and function of each part How is the bacterial agellum different from the eukaryotic one What is chemotaXis In what direction does the agella have to move in order for the cell to show a run instead of a tumble What drives the agellar motion What provides the energy for agella to move What are endospores Why does a microorganism form endospores What triggers the formation of endospores How and why are they formed How do they germinate What are the different reasons proposed on why endospores are so resistant Know the ultra structure of endospore What are the names of the four layers of endospores as seen under electron microscope Which of these layers makes endospores impermeable What is it made up of What makes endospores so resistant Know the various stages in the germination of an endospore into a vegetative cell How would you destroy the endospores of pathogenic organisms Endospores can be looked under the light microscope by simple differential staining Technique Circle the correct answer Endospore germination takes longer time than spore formation Truefalse What are the major differences between Eubacteria Archae and Eukarya Nutrition Growth and Control List the chemical elements needed by the cell in large amounts and in trace amounts What are prototrophs and auxotrophs A mo that requires the same nutrients as most naturally occurring organisms of that species is called an How is it different from an auxotroph Difference between mo based on their nutritional requirement Sources of carbon energy and electrons What are the major nutritional types of mo What are growth factors Name three major classes of growth factors Based on the source of C H and energy which category does each kind of microorganism mo belong to The mo gets energy from oxidation of organic or inorganic compounds The mo gets He39 from organic molecules The mo gets carbon from reduced organic molecules The mo gets He39 from reduced inorganic molecules What is the difference between complex and synthetic medium What is the difference between selective differential and enrichment media What are the different ways for obtaining a pure culture Why would you prefer to pick up cells from the edge of the colony rather than from its center Why do we need solid medium instead of liquid medium to grow bacteria Growth curve What are the 4 phases of growth in a batch culture Explain which of these phases represents balanced growth and which phase represents unbalanced growth What is generation timedoubling time How can you determine the generation time from an organism s growth curve Do microorganisms grow arithmetically or exponentially What is the significance of this Growth pattern Sketch a typical growth curve and brie y describe each of the different phases of the growth curve Which of the following is a reason for the occurrence of a lag phase in a bacterial growth curve The cells may be old and depleted of ATP essential cofactors and ribosomes which must be synthesized before growth can begin The current medium may be different from the previous growth medium therefore the cells must synthesize new enzymes to utilize different nutrients The organisms may have been injured and thereby may require time to recover All of the above are potential reasons for the occurrence of a lag phase Measurement of microbial growth by cell number and cell mass Effect of environmental factors on growth osmolarity pH temp oxygen pressure radiation MO that grow well in salty environment are called MO are classified into different groups based on the optimum temperature at which they grow and the range of temperature that they can tolerate Mention each of these groups How does a psychrophile tolerate low temperatures and how does a thermophile survive at high temperatures Which MO will grow well at 25 0C a Psychrophiles b Mesophile c Thermophiles d Hyperthermophiles
Are you sure you want to buy this material for
You're already Subscribed!
Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'