Regulation of Alternative Sigma Factors

5/24/2018 Regulation of Alternative Sigma Factors

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Regulation of AlternativeSigma Factor Use

Sofia Osterberg, Teresa del Peso-Santos,and Victoria Shingler

Department of Molecular Biology, Umea University, 901 87 Umea, Sweden;email: [email protected]

Annu. Rev. Microbiol. 2011. 65:3755

First published online as a Review in Advance onMay 31, 2011

TheAnnual Review of Microbiologyis online atmicro.annualreviews.org

This articles doi:10.1146/annurev.micro.112408.134219

Copyright c2011 by Annual Reviews.All rights reserved

0066-4227/11/1013-0037$20.00

Keywordstranscription, antisigma factors, ppGpp, DksA, Crl

Abstract

Alternative bacterial sigma factors bind the catalytic core RNA

merase to confer promoter selectivity on the holoenzyme. The difholoenzymes are thus programmed to recognize the distinct pro

classes in the genome to allow coordinated activation of discretof genes needed for adaptive responses. To form the holoenzyme

different sigma factors must be available to compete for their comsubstrate (core RNA polymerase). This review highlights (a) the

of antisigma factors in controlling the availability of alternative factors and (b) the involvement of diverse regulatory molecule

promote the use of alternative sigma factors through subversion domineering housekeeping 70. The latter include the nucleotide

mone ppGpp and small proteins (DksA, Rsd, and Crl), which di

target the transcriptional machinery to mediate their effects.

37

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Core RNApolymerase(core-RNAP): themultisubunit catalyticmachinery of bacterialRNA synthesis

Sigma factor (): adissociable subunit ofbacterial RNAP that

binds with 1:1stoichiometry to core-RNAPs and is essentialfor initiation oftranscription frompromoters by theresulting RNAPholoenzyme

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 38THE TRANSCRIPTION CYCLE

AND DEPLOYMENT OF SIGMAFACTOR DOMAINS . . . . . . . . . . . . . 39

The Sigma Cycle . . . . . . . . . . . . . . . . . . 39

The 70 Family . . . . . . . . . . . . . . . . . . . . 39The 54 Family . . . . . . . . . . . . . . . . . . . . 41

ANTISIGMA FACTORS AND

THEIR ANTAGONISTS . . . . . . . . . 41Counteracting Physicochemical

Assaults . . . . . . . . . . . . . . . . . . . . . . . . 42Responses to Iron Limitation. . . . . . . 42

Partner Switching and Sigma FactorMimicry Mechanisms . . . . . . . . . . . 44

Checkpoint Coupling to Organelle

Biogenesis . . . . . . . . . . . . . . . . . . . . . . 45

PROMOTION OF ALTERNATIVESIGMA FACTOR ACTIVITYTHROUGH SUBVERSION

OF 70 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Alternative Sigma Factors Are Not

Created Equal . . . . . . . . . . . . . . . . . . 46The Bacterial Alarmone ppGpp and

Its Cohort DksA . . . . . . . . . . . . . . . . 46ppGpp and Sigma Factor

Competition . . . . . . . . . . . . . . . . . . . . 47

Modulation of Sigma Factor Usage

Through Diversion of70 . . . . . . . 48CONCLUSIONS . . . . . . . . . . . . . . . . . . . . 49

INTRODUCTION

The temporal and conditional control of tran-scription initiation is a primary access point

for regulating gene expression in all domainsof life. In eubacteria, the evolutionarily con-

served 380-kDa catalytic core RNA poly-merase (core-RNAP; subunit composition con-

sists of2) canaccurately synthesize RNAand terminate transcription at appropriate sites.

However, promoter DNA recognition and ini-tiation of transcription is dependent on a dis-

sociable sixth subunit, namely a sigma factor

(). Association of a given sigma factor with

core-RNAP dictates the DNA-binding spe

ficity of the resulting holoenzyme (-RNAby providing the majority of determinants

recognition of promoter DNA motifs. All bterial species have a housekeeping sigma fac

responsible for transcription from the majity of promoters, and most encode additio

alternative sigmas used to redirect RNAPsets of genes required for adaptive respon(30, 37). Thus, the trademark ability of m

bacteria to adapt to changing ecological coditions is underpinned by highly regulated

namic changes in the functional pools of different-RNAPs that dictate when, and

what extent, the different promoter classesthe genome can be occupied.

Because promoter-binding by a givenRNAP holoenzyme is a prerequisite for c

rect transcriptional initiation, the compositof the holoenzyme pool provides the ba

ground against which other promoter-out

modulating factors must act. These incluclassical DNA-binding transcriptional regu

tors (repressors and activators) and regulatmolecules such as the nucleotide guanos

tetraphosphate (ppGpp) and proteins suchDksA that directly target the active site of

RNAP to modulate its performance at promers (reviewed in Reference 33).

The repertoire of alternative sigma factused to globally alter and coordinate transcr

tional responses to changing cellular dema

varies widely between different species and gerally reflects the lifestyle of the bacterium. F

example, dedicated intracellular pathogens tthrive in a relatively constant environment f

quently possess only a single sigma factor (e

Mycoplasma genitalium). The gut commen

Escherichia colihas 7 sigmas, whereas soil awater bacteria, which are exposed to a pleth

of fluctuating physicochemical and nutritiostresses in their natural environments, poss

many morereaching an excess of a rema

able 60 alternative sigma factors inStreptomycoelicolor (30). The roles of alternative sig

factors in counteracting stress, during biogesis of extracellular appendages, and in devel

mental programs such as spore formation

38 Osterberg del Peso-Santos Shingler

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-RNAP:holoenzyme Rpolymerase

RNAP: RNApolymerase

Guanosinetetraphosphat(ppGpp): the molecule of thstringent respoused here to alencompass ppp

most familiar from studies of the model or-

ganisms E. coli and Bacillus subtilis. However,these represent just a limited subset of the myr-

iad physiological processes controlled by alter-native sigma factors, which extend to pivotal

roles in other development programs, e.g., pro-duction of aerial hyphae byS. coelicolor, regula-

tion of photosynthesis and circadian rhythmsin cyanobacteria, and control of transcrip-tion by bacteria-like RNAP in plant plastids

(30, 37).The ability of sigma factors to capture core-

RNAP to form a holoenzyme is determined bytheir free concentrations and affinity for core-

RNAP. To accommodate the intermittent andenvironment-specific requirement for alterna-

tive sigma factors, bacteria have evolved so-phisticated regulatory systems to control their

production, activity, and availability. In thisreview we first provide a brief overview of the

interactions of sigma factors with core-RNAP

and promoters as a preface to highlighting howthese critical interfaces are exploited by anti-

sigma factors. Second, we focus on mechanismsby which targeting of the housekeeping sigma

and/or its holoenzyme by regulatory moleculescan provide a more general strategy to promote

the use of many alternative sigma factors.

THE TRANSCRIPTION CYCLEAND DEPLOYMENT OF SIGMAFACTOR DOMAINS

The Sigma Cycle

Within the holoenzyme, the sigma factor makes

multiple and extensive contacts with core-RNAP and plays an active role in initial pro-

moter engagement to form a closed promotercomplex and in subsequent steps of DNA melt-

ing to form the open promoter complex re-quired for transcriptional initiation (Figure 1).

Tight-core-RNAP association is sequentiallybroken prior to promoter escape of RNAP into

the elongation mode (66). However, complete

detachment of the sigma is not a prerequi-site for transcriptional elongation per se, and

a partially attached sigma can cause elongation

Initiation Elongation

a

Stochastic release

Co

Promoterengagement

Sigma binding

Termina

Competition

b

R+P RPc RPi RPo

NTPs

RPinit

NTPs

RPE

Figure 1

The sigma cycle allows reprogramming of core RNA polymerase(core-RNAP). (a) Schematic illustration of the transcription cycle in whsigma factors compete for association with core-RNAP to direct theholoenzyme to engage promoters. (b) Simplified schematic of the multireversible steps of transcriptional initiation: -RNAP (R) binds promot(P) to form the initial closed complex (RPc), which, through sigma-assiformation of a number of unstable intermediate complexes (RPi), evenleads to the open complex (RPo), which is competent to initiate transcrNote that reversible steps of initiation end as the elongation complex (R

escapes the promoter.

stalling by binding promoter-element mim-ics within DNA. Nevertheless, the majority of

sigma factors are rapidly, albeit stochastically,released during elongation (65, 74) to join the

pool of free sigma for competitive associationwith core-RNAP. The release of sigma during

each round of transcription provides the cen-

tral mechanism for reprogramming the levelsof the alternative -RNAP holoenzyme pools

and thus cognate promoter occupancy.

The70 Family

With the exception of homologs of E. coli

54 (see below), all alternative sigmas belong

to the extensive 70 family, named after the

www.annualreviews.org Control of Sigma Factor Use 39

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54

70

a

b

4 3 2 1.1

3.2

DNAmelting

Corebinding

DNA-bindinginhibition

3.2 loop

Core binding bEBPinteractions

DNA interactions

24 12

NCR

UP element 35 Ext. 10 10 Discriminator

RpoN box

TTGACA TGn TATAAT GGGnnn

4.2

4.1

3.1

3.0

2.4

2.3

2.2

2.1

1.2

C N

C N1.1

IIIRegion III

TTGGCACG TTGC

Figure 2

Sigma factor domains and their functions. (a) For 70 proteins, roles of theconserved subregions within the 2, 3, and 4globular domains as describedin the text are highlighted. NCR indicates the location of a nonconservedregion. Consensus for the 35 hexamer (35 to 30), the extended 10element (Ext.; 15 to 13), the 10 hexamer (12 to 7), and discriminatorDNA (6 to 1, with an optimal GGG6 to 4), relative to thetranscriptional+1 start, are taken from References 33 and 56. (b) For 54

proteins, consensus for the 24 (27 to 20) and 12 (15 to 12) elements,which encompass almost invariant GG and GC recognition motifs(underlined), are taken from Reference 78. Abbreviation: bEBP, bacterialenhancer-binding protein.

Antisigma factor:any agonist that

through binding to asigma factor inhibitsits ability to associatewith core-RNAP

ECF:extracytoplasmicfunction

housekeeping70 ofE. coli(also known as D;

A inB. subtilisand many other species). A dual

(or sometimes triple) naming system for E. colisigmas is prevalent in the current literature;

therefore, after their first introduction we useonly numerical or gene name superscripting for

E. colisigmas.Extensive biochemical, genetic, and struc-

tural analysis has underscored the roles ofdifferent domains of housekeeping sigmas

in providing four of the five known inter-actions that occur with the promoter DNA

(Figure 2a). At some promoters, a fifth

interaction is provided by the -subunitsof core-RNAP at an AT-rich UP-element

DNA (reviewed in Reference 33). The most

conspicuous 70-promoter recognition e

ments are the 35 and 10 hexamers that contacted by the 4 and 2 domains of respectively (17, 66). Subregion 1.2 within

2domain can also provide promoter conta

through the discriminator DNA downstre

of the 10 element (34). At extended

promoters, which frequently lack a discerna35 element, additional promoter contacts provided by interaction between the3dom

and DNA just upstream of the 10 elemennonconserved region of highly variable len

intersperses the 2 domain of some houkeeping sigmas. ForE. coli70, this region

been implicated in assisting dissociation of sigma factor to alleviate pausing during

early stages of elongation (54).

The 70 family of proteins is divided i

subgroups based on phylogenic relations adifferential possessionof thefour conservedmains (2, 3, 4, and region 1.1) (Figure

(reviewed in References 30 and 68). Groucomprises housekeeping sigmas that possess

four domains, including the group-specific gion 1.1, which is involved in autoinhibition

DNA binding by free sigmas. Group 2 sigmrepresented by theE. colistationary/stress f

tor 38/S, are related most closely to Groubut are dispensable for growth. Group 3 s

mas, which are more distantly related to Gro

1 (e.g., E. coli 28/F/FliA and 32/H; B. sub

F), also possess all three globular domains a

usually control regulons in response to devopmental checkpoints or heat shock. The

domains of E. coli 28 and 32 interact wcomposite extended10/10 elements that

count for the unusually long consensus recnition elements of their cognate promoters (

51). The final and most divergent group of smas is Group 4the extracytoplasmic funct

(ECF) subfamily, so named because most spond to signals arising from the extracytopl

mic environment (e.g., E. coli24/E and F

Group 4 represents the most stripped-doversion of sigmas, possessing the only two

sigma domains (2and 4) that are structurconserved even among the most divergent fa

ily members.



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The54 Family

This second class of sigma factors is uniquely

represented by orthologs ofE. coli54/N, whichdirects recognition of distinct promoter motifs

located at positions 24 and 12 relative tothe transcriptional start (Figure 2b). Although

initially identified for their role in nitrogen

assimilation, 54 proteins are widely distributedin bacteria and are utilized in coordinatingmany different physiological processes ranging

from utilization of alternative carbon sources,through assembly of motility organs, to pro-

duction of extracellular alginate. Although 54

proteins need to perform many of the same

functions as other sigmas, they bear no primary

sequence similarity to70 proteins and regulatetranscription by a different mechanism. A key

feature of 54-RNAP that contrasts other

holoenzymes is its complete inability to spon-taneously isomerize (melt) DNA to form openpromoter complexes. This step strictly re-

quires assistance from mechanotranscriptionalactivators (also known as bacterial enhancer

binding proteins, or bEBPs) that utilize ATPhydrolysis to drive conformational changes for

this transition (reviewed in Reference 78).Genetic and biochemical data on the roles

of the three main subregions of 54 (regions

I to III, Figure 2b) have recently been aug-

mented by structural analysis and cryoelectronmicroscopy reconstructions (12, 42). Region Imediates weak contact with the 12 promoter

element and with core-RNAP such that itoccludes loading of promoter DNA into the

active site. The varying region II links regions Iand III, which makes the main contacts with the

24and12 promoter elements andwith core-RNAP (Figure 2b). Activation by an obligatory

activator serves two functions. First, relocating

region I from an inhibitory conformation al-

lows entrance of the DNA. Second, facilitatingcorrect promoter DNA-54 alignment allowsfor open complex formation (12). Because of

the unique properties imparted by54 (e.g., un-usual promoter recognition, the ability to bind

DNA in the absence of core-RNAP, and the ne-cessity for ATP-utilizing mechanoactivators),

54-dependent transcription is considered a

second paradigm of bacterial transcription.Although the 54 and the 70 family members

lack sequence identity, they do bind overlap-ping surfaces of core-RNAP, and performance

of 54-RNAP is affected by the same mobile

modules of the core-RNAP - and -subunits

that influence 70

-dependent transcription,albeit with different regulatory outcomes (21,88). Hence, 54 is not exempt from funda-

mental regulatory mechanisms that involvecompetitive association with core-RNAP.

ANTISIGMA FACTORS ANDTHEIR ANTAGONISTS

Modulating the levels and/or activities of differ-

ent sigma factors, and consequently the levels

of cognate RNAP holoenzymes, provides a sim-ple yet versatile means to control the basal-line

occupancy of distinct promoter classes. Mech-anisms known to modulate the activities of sig-

mas are diverse and include phosphorylation-activated binding to a partner protein that

tags the sigma for destruction (e.g., interac-tion between the E. coli response regulator

RssB and 38) (89), proteolytic cleavage ofinactive prosigmas to remove inhibitory N-

terminal extensions (e.g.,B. subtilisproE and

proK) (41), and signal-cued use of alterna-tive start codons to generate high-molecular-

weight variants that are vulnerable to rapidproteolytic turnover (e.g., S. coelicolorR and

itsMycobacteriumortholog) (49). For some sig-mas, protein levels are controlled at all steps of

gene expressionfrom transcription initiation,through mRNA stability and control of transla-

tional efficiency by small noncoding RNAs, tosignal-responsive proteolytic degradation (e.g.,

E. coli38 and32) (31, 38). In many other cases,

however, the primary level of control involvessequestering by antisigma factors to preventtheir association with core-RNAP. The follow-

ing sections providean overview of selected sig-

mas that illustrate different systems that controlthe release of sigma factor activities when they

are needed.


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Coantisigma factor:a factor that acts inconcert with a partnerto bind and sequester asigma factor andthereby prevent

association withcore-RNAP

Counteracting PhysicochemicalAssaults

The Group 4 ECF sigmas encompass 60% ofall sigma factors in bacteria (15) and are chiefly

associatedwith counteracting physical or chem-

ical stresses or communicating the availabilityof iron. A common feature of most ECFs is

that their activity is regulated by stoichiomet-ric association with an antisigma factor, which

is usually coexpressed through transcriptionalcoupling of the genes within an operon. This is

the case forE. coliECF 24, which controls re-sponses to membrane stress and represents one

of the rare exceptions to the dispensable natureof alternative sigma factors (23).

The 24 gene (rpoE) is cotranscribed

with those of its antisigma factor RseA andits coantisigma factor RseB, which tightly

tether 24 to the membrane in an inactive state(Figure 3a). Themolecular details of the three-

compartment proteolytic cascade that governs

24 availability have previously been extensively

reviewed(4,14,36),soonlyanoverviewisgivenhere. The DegS serine protease is both the

molecular sensor of stress-induced misfoldedproteins within the periplasm and the initiator

of the RIP (regulated intramembrane proteol-ysis) cascade that releases 24. Activation of the

proteolytic activity of DegS results in cleavage

of the antisigma RseA within its periplasmicregion (site 1 cleavage), rendering it as a

substrate for the metalloprotease RseP, whichin turn processes RseA within its inner mem-

brane spanning region (site 2 cleavage). Thereleased cytoplamsic RseA/24 subcomplex still

sequesters 24 but has an exposed tag for recog-nition by the adaptor protein SspB that directs

the complex to ClpXP for final processing tofree 24 for association with core-RNAP.

Similar RIP cascades likely control the avail-

ability of gram-negative 24

orthologs andother ECFs such as AlgU, which is involved

in the production of alginate byPseudomonasaeruginosa with devastating consequences for

cystic fibrosis patients (reviewed in Reference36). Likewise, although gram-positive bacte-

ria lack a periplasm and the mechanistic details

differ, a conceptually similar RIP cascade co

trols the stress responses mediated byB. s

tilisW that is cotranscribed with its antisig

RsiW (36). Sequestering of a sigma to the mebrane is an efficient means to couple availab

ity to extracellular signals or those that resin alterations within the periplasmic compa

ment. A few ECFs, however, govern responto intracellular stress and are consequently ctrolled by cytoplasmic antisigma factors. T

is the case for theS. coelicolorR/RsrA (48) atheRhodobacter sphaeroidesE/ChrR (6) syste

that control responses to damaging oxygspecies.

Irrespective of the cellular location, efficisequestering requires the sigma/antisigma

teractions to be tight and mask portionskey interfaces usually involved in interact

with core-RNAP (19). The cytoplasmic ption ofE. coliRseA has been estimated to b

E with 300-fold-higher affinity than co

RNAP. Structural analysis has shown that questering involves the N-terminal domain

RseA, which sterically occludes the criticaland 4domains of this ECF (18). Despite li

primary sequence homology, an analogous main with a common fold within ChrR likew

exploits the same interfaces in its interactiowith E of R. sphaeroides (15). Bioinform

searches indicate that the common domainRseA and ChrR (ASD, antisigma domain

fused to diverse signaling domains in >30%

all ECFs (i.e.,20% of all annotated sigma ftor genes). Thus, manipulation of the geome

ofthecritical2and4domains likely undersequestering and release by cognate antisig

factors in many systems.

Responses to Iron Limitation

In addition to stress responses, ECFs are a

frequently involved in processes that ensa sufficiency of iron, which is often in li

ited environmental supply. For example,

Pseudomonas putida, 13 of its 19 ECFs apear to be dedicated to this essential elem

(61). This subgroup of ECFs is usually transcribed with a cognate antisigma factor



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Sequestered

P

P

P

a

b c

Cytoplasm

Innernnermembraneembrane

Inner

membrane

Periplasm

Outerutermembraneembrane

Outermembrane

Stress

DegS

OmpC RseBRseB

RseA

24/E

RseP

OmpC*

SspB

ClpXP

Available

PhyR

NepR

EcfG1

Sequestered

Stress

Available

Basal body

FlgM

Sequestered

28/FliA

Available

Hook

mimicdomain

1

2

3

Figure 3

Control of sigma factor availability by antisigma factors. (a) Schematic illustration of DegS/RseP RIP(regulated intramembrane proteolysis) protease cascade (allblue elements) that releases 24/E fromsequestration at the membrane by its antisigma factor RseA and coantisigma factor RseB. Activity of the RIPcascade is triggered by stress that elicits misfolded proteins in the periplasm (such as OmpC ) to eventuallyrelease a RseA/24 subcomplex that is guided to the cytosolic ClpXP protease by SspB for final trimming torelease 24 to compete for core RNA polymerase (core-RNAP). The sequential cleavage sites (1 to 3) withinRseA are indicated. (b) The antisigma factor NepR sequesters ECfG1 within the cytoplasm until stresssignals result in the phosphorylation of the receiver domain of the anti-antisigma factor PhyR.Phosphorylation of PhyR exposes a sigma mimic domain of PhyR that recruits and sequesters the antisigma(NepR), thus leaving ECfG1 free to associate with core-RNAP. (c) The antisigma factor FlgM likewisesequestersFliAwithin the cytosol. However, in this instance, completion of the flagella basal body allowsexport of partially unstructured FlgM, resulting in a pool of available 28 ready for holoenzyme formation.

control expression of genes involved in theuptake of iron-scavenging siderophoresfirst

through an outer membrane siderophore trans-porter and then from the periplasm to the cy-

tosol via an ABC-type transporter. The mostextensively studied siderophore signaling path-

way is that of the Fec system for ferric citrate

uptake inE. coli, which represents a paradigmsystem for responses to a signal that can only

enter the cell through transport.Signaling involves communication from

the cell surface siderophore transporter (FecA)to the inner-membrane-anchored antisigma

factor (FecR), which sequesters FecI to the



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Anti-antisigmafactor: a factor thatantagonizes orcounteracts the activityof an antisigma factor

cytoplasmic side of the inner membrane. Cell

surface binding of ferric citrate to the FecAtransporter triggers structural changes that

facilitate interaction between the N-proximalportion of FecA and the C-terminal portion

of the antisigma FecR within the periplasm.FecR then transmits the signal across the inner

membrane to its N-terminal portion to relieveinhibition of FecI activity in the cytoplasmcompartment. The regions of FecA that are

involved in transducing signals arising fromsiderophore transport are comparatively well

understood; however, the details of the mecha-nisms that underlie thepropagation of the ferric

citrate-binding signal to the C-terminalportionof FecR, and from there through FecR to affect

FecI activity, remain an open question (14).In some Fec-like systems the antisigma fac-

tor only has a negative effect on sigma activ-ity and, upon receiving the activating signal,

may well simply release the sigma for asso-

ciation with core-RNAP, as is typical of an-tisigma factors. However, for FecI/FecR and

some other related systems, activation throughthe FecA transporter converts the antisigma

factor from a negative to a positive regulatorthat stimulates the activity of the cognate sigma

factor (63 and references therein). In thesecases, the antisigma likely remains bound to the

sigma to achieve this outcome. Genetic analysiswith truncates and point mutations ofFecI has

demonstrated that FecR sequesters FecI only

via interaction with its 4domain, and that thisinteraction is required for FecI to function as

a sigma factor (60). Sequestering solely via the

4 domain may be a reflection of the relative

unimportance of the 35 element (that is usu-ally bound by a 4domain) for FecI-dependent

transcription (5). Rather than FecI release, sig-naling to the cytoplasmic N terminus of FecR

stimulates FecI association with the -subunitof RNAP (59). Because portions of FecR, FecI,

and stably interact simultaneously (59), andactivation via FecR also promotes novel pro-

moter interactions with DNA at +13 fromthe transcriptional start site (5), the activation

model that emerges is one in which FecR re-

mains a functional part of the transcriptional

initiation complex. Tethering to the in

membrane via FecR would not necessarily ipose a hindrance to transcriptional elongat

because the sigma factor is released duringshortly after) transcriptional initiation. Ho

ever, it does pose the interesting conundrof how the FecI-RNAP holoenzyme locate

target promoter from its restricted location

Partner Switching and Sigma FactorMimicry Mechanisms

Bacillus species provide prime examplescascade production and compartmentalizat

of sigma factors, as well as other paradigof how the activities of alternative sigm

can be controlled. Both the F forespdevelopmental program and the expression

the B stress regulon ofB. subtilisare governby analogous phosphorylation-depend

partner switch mechanisms (reviewed

References 35 and 41). In these systeswitching between alternative binding partn

of the antisigma factorfrom the sigma facto an anti-antisigma factoris the crit

step that releases the sigma to performfunction. The activity of F is regulated

the serine kinase antisigma factor SpoIIAB athe anti-antisigma factor SpoIIAA, which

cotranscribed with the F gene (sigF) in

spoIIAoperon. Structural determinations h

highlighted the importance of the3domain

F for sequestration by its antisigma (SpoIIA(16). Dimeric SpoIIAB binds asymmetrica

to a single molecule ofF and occludes itsdomain from interaction with core-RNA

As a result of the asymmetric binding, onethe SpoIIAB protomers is more accessible

binding to the anti-antisigma factor (SpoIIAthat, upon docking to SpoIIAB, displaces

to leave it free to associate with core-RNASpoIIAB then phosphorylates its new part

SpoIIAA in a reaction that dissociates ADP-bound form of SpoIIAB, which in t

associates with any unphosphorylated SpoIIto form a complex that inhibits both the kin

and antisigma activity of SpoIIAB (41, 62, a

references therein).



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Binding partner switching is also an in-

tegral component of the general stress re-sponse in Alphaproteobacteria that lack an

E. coli38 or B. subtilisB ortholog. However, inthese organisms, control involves the unusual

anti-antisigma PhyR, which has a C-terminalresponse-regulator (RR) domain coupled to

an ECF-like domain. Upon phosphorylation,Methylobacterium extorquensPhyR binds NepR,an antisigma factor that normally sequesters

the ECF EcfG1 (Figure 3b) (26). Although theECF-like domain of PhyR shares high homol-

ogy with EcfG1, it lacks critical residues thatwould be involved in DNA binding (82) and

thus appears to serve as a pure mimic to en-tice NepR away to free EcfG1 to serve its du-

ties. A recent structure of Caulobacter crescen-

tusPhyR in its unphosphorylated state shows

predictable structures for its component parts.However, extensive interactions between the

RR domain and the ECF domain (2linked to

4) force the 2and 4modules into a compactconformation that presumably prevents recog-

nition by NepR until PhyR is phosphorylated(40). Based on the distinct structural compo-

nents, and the fact that the ECF domain alonecan act as an anti-antisigma, the authors pro-

pose that the RR domain can be considered asan antianti-antisigma factor. The counterpart

sensor kinase(s) (or anti-antianti-antisigma fac-tor) that would serve to phosphorylate PhyR,

and thus initiate the whole cascade, remains to

be identified. However, a variety of candidateperiplasmic or cytoplasmic sensor kinases are

encoded in the vicinity ofphyRgenes in differ-ent organisms (81).

Checkpoint Couplingto Organelle Biogenesis

Flagella are characteristically assembled ina stepwise manner through temporally con-

trolled expression of their component parts.The hierarchical expression of flagella genes

can be achieved by diverse mechanisms but usu-ally involves a master regulator that initiates the

cascade and coupling of late flagella gene ex-

pression to completion of the hook basal body

structure to form a developmental checkpoint

(79). In most flagellated bacteria, the key play-ers in this developmental checkpoint are an an-

tisigma factor (FlgM) and a specific sigma fac-tor (E. coliFliA,B. subtilisD) that is required

for transcription of genes encoding the flagellafilament subunits and proteins involved in bac-

terial taxis. Upon completion of the hook basalbody structure, FlgM can be secreted throughthe type III system housed within the basal

body, thus releasing FliA for association withcore-RNAP (Figure 3c). Hence, this mecha-

nism uses secretion as a signal that cues com-pletion of a functionalactive structure to ensure

that filament and taxis proteins are expressedonly when appropriate (43).

NMR studies have established that FlgMis intrinsically partially disordered, with only

the C-terminal half structured when in associ-ation with FliA and under molecular crowding

conditions that would prevail in vivo (22, 24).

The naturally unfolded state of FlgM (com-plete or partial) has been suggested to facili-

tate secretion of FlgM through thenarrowhookbasal body structure (22). Biochemical and ge-

netic evidence that implicates multiple regionsofFliA in its sequestration by the C-terminal

of FlgM hasbeen reinforced by crystallographicdata from theAquifex aeolicusFliA/FlgM com-

plex, which shows a highly compact conforma-tionofthe2,3,and4 domainsofFliA,which

masks its DNA-binding and core-RNAP asso-

ciation determinants (80).The FlgM and FliA genes are not encoded

within an operon, rather the levels of FlgMare frequently under complex regulation that

includes (a) dual promoter control of theflgMgene, in which one promoter isFliA dependent

and thus provides an autorepressing feedbackcircuitry; and (b) translational modulation of

FlgM protein levels, which adjusts the relativeFlgM:FliA ratios within the cell (reviewed in

Reference 79). Recently, mathematic modelingand experimental rewiring of the flgM and

fliA gene promoters have provided strongsupport for a previously proposed model

in which FlgM secretion, in addition to

enforcing the developmental checkpoint,



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functions as a proxy-measuring system that

continually fine-tunes FlgM and FliA levelsto provide a sensing mechanism that may also

control flagella numbers (77).

PROMOTION OF ALTERNATIVESIGMA FACTOR ACTIVITY

THROUGH SUBVERSION OF70

Alternative Sigma Factors Are NotCreated Equal

As highlighted in Figure 1, active transcrip-tion and the sigma cycle provide the means

for reconstituting alternative -RNAP holoen-zymes. However, as exemplified by the find-

ings inE. coli, alternative sigmas generally havelower affinity for core-RNAP than the house-

keeping 70, with the poorest (38) estimatedto be approximately 10-fold lower (57). More-

over, even when presented with conditions that

maximize the levels of active alternative sig-mas, the cellular concentrations of alternative

sigmas are greatly exceeded by those of 70

(29, 72, 75). The levels ofE. coli70 and core-

RNAP are relatively constant over the growthcurve and under different growth conditions.

Although absolute values differ somewhat, thenumber of70 molecules is consistently esti-

mated to exceed that of core-RNAP by approx-imately threefold (29, 72, 75). Because much of

the core-RNAP is employed in catalyzing RNA

synthesis, competition betweensigmafactors toform a holoenzyme with the limited free core-

RNAP will be fierce.Many approaches, including artificial ma-

nipulations of sigma factor levels and the useof sigma factor mutants that are altered in

their affinity, and thus their competitiveness,for core-RNAP, have clearly established that

sigma factor competition for core-RNAP lim-its output from promoters dependent on al-

ternative sigmas such as 38, 32, and 54 (46,53). This is likely the case for all alternative

sigmas, which raises the question of how low-level and/or weak-affinity alternative sigma fac-

tors gain sufficient access to core-RNAP to

drive transcription from promoters under their

control. The following sections present e

dence that global regulatory subversion of household 70 to concomitantly enhance f

mation of alternative-RNAPsprovidesatlea partial solution to the problem.

The Bacterial Alarmone ppGpp

and Its Cohort DksA

The nucleotide ppGpp (also known as ma

spot) is the primary mediator of the stringresponse to amino acid starvation, wh

translational capacity is balanced to redudemand through downregulation of transcr

tion from tRNA and rRNA operon promot(stringent70 promoters). In addition to am

acid starvation, iron, carbon, and nitrolimitations, as well as many environmen

physicochemical stresses that reduce growrate, cause induction of the intracellular lev

of ppGpp (73). The rapid elevation of ppG

levels during the hungry phase (just prior totransition between exponential and station

growth in rich media), or through artifimanipulation of ppGpp levels under norm

nonpermissive conditions, markedly enhanoutput from many promoters dependent

alternative sigmas (e.g.,E. coli38, 32, 24, a

54) (20, 28, 46, 53, 84). It is now evident t

ppGpp is the mediator of a far greater netwthat holistically redirects the global transcr

tional capacity of the cell from genes for grow

toward those for adaptive survival respon(32, 73).

Targeting of the -RNAP by ppGpp speically alters its performance at susceptible p

moters to either decrease (e.g., stringentpromoters) or increase (e.g., some 70 p

moters involved in amino acid transport orvirulence, and some specific 24- and F

dependent promoters) their activities (2, 47, 67, 70, 71). The exact location and l

anding residues for ppGpp within the tive site cleft of core-RNAP remain elus

(86). Nevertheless, it appears clear that ppGbinding to RNAP lowers the energy requi

for transition between intermediates in

pathway leading to open-complex formati



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Depending on the rate-limiting step and rel-

ative stabilities of consecutive intermediates,lowering the energy required for conversion

from one intermediate to the next would fa-vor either the reverse or forward reactions in

Figure 1b, leading to promoter-specific nega-tive or positive outcomes (reviewed in Refer-

ence 33).The in vivo and in vitro effects of ppGpp atpromoters are frequently amplified by DksA

a member of a family of regulators that bindsRNAP and accesses the active site through the

secondary channel. DksA mediates long-rangestructural changes within RNAP that alter in-

teraction with the 6 to +6 region at70 pro-moters (11, 55, 76). Although it remains to be

experimentally tested in most cases, there is noreason why DksA could not also affect the per-

formance of any-RNAP, although the con-sequences might differ. DksA and ppGpp can

have mutually independent and sometimes op-

posing effects (1, 3, 75, and references therein);however, DksA sensitizes RNAP to the cellular

levels of ppGpp to account for their commoncoaction (70). InE. coliandP. putida, DksA lev-

els are relatively constant (9, 75); therefore, itis the changing levels of ppGppthe herald

of stressthat instigate proactive promoter-specific and global transcriptional responses

that allow the cell to prepare for tough timesahead.

ppGpp and Sigma Factor Competition

As a global regulator, ppGpp by definition haspleiotropic effects in vivo, and thus many dif-

ferent mechanisms frequently converge to ulti-mately account for the total effect of ppGpp on

output from a given promoter. These includepromoter-specific effects on the performance of

holoenzymes at kinetically susceptible promot-ers (see above), which in turn can initiate ad-

ditional regulatory cascades through the geneproducts they encode. However, these regula-

tory consequences cannot account for the full

effect of ppGpp in vivo. For example, ppGppaids stability ofS through production of anti-

adaptors to result in higher cellular levels ofS

under stress conditions (reviewed in Reference

8). Nevertheless, aS promoter that is not de-pendent directly on ppGpp still requires ppGpp

for activity in vivo even when reduced S lev-els are compensated for by ectopic expression

(46, 52). Likewise, although the levels of54

are constant irrespective of the presence or ab-

sence of ppGpp and/or DksA, the activities of54 promoters that are not enhanced directly byeither factor in vitro are still greatly stimulated

bythepresenceofthesemoleculesinvivo(9,10,53, 84). These findings demand an alternative

explanation for the action of ppGpp.In E. coli, elevated intracellular levels of

ppGpp result in decreased association of 70

and core-RNAP (but not decreased 70 lev-

els per se), so that less 70-RNAP is avail-able to occupy cognate 70 promoters (32, 39).

In addition, a proteomic approach has shownthat underproduction of70-RNAP essentially

mimics the stringent response (58). Separa-

tion and immunological detection of free andcore-RNAP-associated sigma has been used to

demonstrate that elevated ppGpp, which de-creases70-RNAP levels, concomitantly results

in increased38-RNAP and32-RNAP holoen-zyme levels (46). Although not experimentally

tested, this would be anticipated to also bethe case for other -RNAPs. These data, to-

gether with the finding that the requirementfor ppGpp to achieve efficient 38- and 54-

dependent transcription can be simplybypassed

using 70 mutants that are defective in theirability to compete for binding to core-RNAP

(10, 46, 53, 84), make a convincing case forthe idea that ppGpp plays a determining role

in the outcome of sigma factor competition tofavor holoenzyme formation with alternative

sigmas.Whereas the importance of ppGpp in vivo

is clear, it remains to be resolved if ppGppand/or DksA can actively alter sigma factor

competition, or if their effects occur indirectly(passively) through 70-dependent transcrip-

tion. An active role has been suggested basedon the observation that ppGpp enhanced tran-

scription from a 32-dependent promoter only

under conditions of competition with 70 in



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vitro (46). However, such stimulation was not

found in similar experiments with54 in com-petition with 70, neither in the presence nor

in the absence of ppGpp and/or DksA (10, 53,83). In addition to altering sigma factor compe-

tition, 70- and core-RNAP - and -bypassmutants also functionally mimic the action of

ppGpp and DksA by further destabilizing thenotoriously unstable open complexes of rRNAoperon promoters (7, 83). The latter property

suggests a passive mechanism by which theseregulatory molecules could alter sigma factor

competition.A unifying model has been proposed that

would explain the properties of bypass mutantsand enhanced performance by any alternative

sigma in the presence of ppGpp (10, 83). Thismodel, like many before it, invokes passive

regulation through the consequences of thenegative action of ppGpp and DksA at the

seven powerful stringent 70-rRNA operon

promoters. InE. coli, the activities of these70

promoters sequester approximately 60%70%

of the transcriptional machinery during rapidgrowth in rich media (13). Under these con-

ditions, where ppGpp levels are low, much ofthe core-RNAP is occupied in catalysis of the

transcripts from these powerful promoters,leaving little available for association with any

sigma factor. This would lead to low levelsof alternative holoenzymes and consequent

low occupancy and output from promoters

under their control. Under slow growth and/orstress conditions that elicit high levels of

ppGpp, however, the potent downregulationof transcription from the 70-rRNA operon

promoters would lead to increased levels ofcore-RNAP available for holoenzyme forma-

tion. As a consequence, alternative -RNAPlevels would increase even in the absence of

a change in sigma levels, leading to enhancedpromoter output from cognate promoters.

Within this model, decreased 70 availabilityand 70-RNAP mutants would mimic high lev-

elsofppGppbyreducingtranscriptionfromthepowerful stringent70-rRNA operon promot-

ers and by altering core-RNAP to sigma factor

association to favor alternative holoenzyme

formation. A prediction from this model is t

low-affinity promoters that have holoenzybinding as a rate-limiting step would be m

susceptible to loss of these regulatory molecuthan high-affinity counterpartsa predict

that has been experimentally verified to be case for 54-dependent transcription (9, 10)

Modulation of Sigma Factor UsageThrough Diversion of70

The model outlined above does not exclu

nor is it incompatible with, the possible extence of unknown factor(s) that may additi

ally contribute to the in vivo requirement ppGpp and DksA. Analogous to the discov

of the role of DksA in ppGpp-mediated rulation, it cannot be ruled out that some ot

protein(s) may facilitate ppGpp-mediated relation of alternative-RNAPs or aid their f

mation in vivo. On the contrary, the ppG

triggered reduction of 70-RNAP levels (not those of 70 itself) demands that 70

diverted to prevent its association with coRNAP. The answer to how this is achieve

not currently fully understood. However, asscribed below, the Rsd (regulator of sigma

protein is likely a major contributor, and ftors such as the Crl protein are also potentia

involved.Rsd was initially identified through a sea

for factors that might allow alternative sig

factors to compete for limiting core-RNAPE. coli(45). By forming 1:1 complexes with Rsd specifically sequesters free 70 and can aactively remove 70 from 70-RNAP in vi

(44, 87). Biochemical and genetic studies of Rand its AlgQ homolog have shown that th

proteins sequester 70 primarily through teractions with the4 domain (which conta

35 elements), although other contact poiwith 70-RNAP are also involved (25, 4

Structural studies of Rsd in complex withof70 have revealed that Rsd binding occlu

residues critical for 4/core-RNA interacti(69). By binding 70 and occluding associat

with core-RNAP, Rsd falls under the definit

of an antisigma factor. However, this definit



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seems inappropriate because Rsd can be vastly

overexpressed without compromising growth.Transcription of the Rsd gene is directed

by the activities of two promoters, and is par-tially under ppGpp control, leading to ele-

vated levels of Rsd when competition for core-RNAP would be at its highest (45, 72). The idea

that Rsd might facilitate formation of alterna-tive holoenzymes via reducing the availabilityof 70 has been spurred by the findings that

overexpression of Rsd results in increased out-put from some 54-, 38-, and 32-dependent

promoters (46, 53, 64), and because additionof Rsd can facilitate sigma factor exchange in

vitro (L. Holmfeld & V. Shingler, unpublisheddata). Consistent with the idea that Rsd could

facilitate access of alternative sigmas to core-RNAP, the naturally elevated levels of Rsd in

stationary-phaseE. colisequester a significantportion (25%) of70 (72). However, it should

be emphasized that Rsd null mutants have min-

imal effects on 38- and 54-dependent pro-moter outputs that are enhanced by overexpres-

sion of Rsd (9, 64), suggesting that Rsd doesnot act alone to bring about these regulatory

events. An intriguing finding from structuralstudies of the Rsd/4 complex is that a network

of interactions connects the binding interfacewith other potential binding cavities located on

the surface of Rsd. Although some of these in-teractions may be involved in recognition of

70-RNAP, this observation raises the possibil-

ity of functional coupling of Rsd/70 bindingwith binding of some as yet unknown protein

and/or small regulatory molecule (69). If this isindeed the case, identification of such an entity

would surely further our understanding of thephysiological role of Rsd.

TheE. coliCrl protein binds 38 and pref-erentially favors 38 in competition with 70

for core-RNAP, presumably by facilitating38-RNAP holoenzyme formation (27, 85). The Crl

protein is restricted in its genome distributionand the global regulatory effect of Crl is lim-

ited to promoters of the 38 regulon (85). Thismakes it unlikely that Crl has any significant

effects on the levels of other -RNAP holoen-

zymes. Nevertheless, it does pose an alternative

scenario to specific 70 sequestration to at least

partially account for reciprocal alterations in

70-RNAP versus alternative-RNAP holoen-

zyme levels. Given the large number of genes ofunknown function inE. coliand other bacteria,

it is certainly plausible that analogous facilita-

tors of other alternative holoenzymes exist but

have eluded detection.

CONCLUSIONS

Regulation of alternative sigma factor activity is

usually complex, with multiple tiers of controlto regulate both their expressionlevelsandtheir

activities. One major mechanism is sequester-ing by an antisigma factor, which provides sys-

tems for signal-specific control of sigma fac-tor availability and thus the activity of promot-

ers they regulate. Where known in any depth,these systems areexquisitely attuned to both the

typeofsignal(i.e.,thecompartmentfromwhich

thesignalarises)and the constraints imposed bythe nature of the signal (e.g., the need for iron

transport into the cell). However, for many al-ternative sigmas, particularly those of the ex-

tensive ECF Group 4 family, only a few havebeen studiedin any detail. In many cases neither

the signal nor the factor(s) that controls theiractivity is known, which severely curtails un-

derstanding their role in microbial physiology.Given the novel mechanisms that have recently

been identified by studying new members of

this familysuch as the use of alternative startcodons to generate proteolytically vulnerable

variants of S. coelicolor R (49) and molecu-lar mimicry of EcfG1 in Alphaproteobacteria

(26)it is not unreasonable to expect the reper-toire of mechanisms that can control sigma fac-

tor availability to continue to expand.Sequestering by an antisigma factor both

protects the sigma factor from proteolytic at-tack and provides immediate availability upon

demand. Genetic and structural studies ofsigma/antisigma interactions have identified

repeated themes within sequestering mecha-

nisms, namely manipulation of the geometryof the key globular sigma domains (2, 3,

and/or4) to mask critical regions involved in



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interaction with core-RNAP and promoter

DNA. Likewise, similar studies have alsostarted to unravel and test models of how

antisigma/anti-antisigma interactions breakthese interactions to free sigmas to perform

their function.In addition to dedicated signaling pathways,

the activities of manyE. colialternative sigmafactors can be coordinately stimulated by theglobal regulator ppGpp, and this is likely to also

be the case in other organisms. The adoption

of ppGpp to stimulate the activities of alter

tive sigmas is perhaps not surprising becastresses that elicit ppGpp synthesis over

greatly with those that cue the need for altnative sigmas. Much evidence has accumula

that this stimulatory effect occursthrough a r

of ppGpp in determining the outcome of sig

factor competition for limiting core-RNto holistically favor association of alternatsigmas over 70. However, much remains to

learned about how this is brought about.

SUMMARY POINTS

1. The transcription cycle provides the means to rapidly strip off the sigma factor to gen-

erate naked core-RNAP ready for reprogramming by any available sigma. However, theactivities and availability of most sigma factors are intricately controlled.

2. Antisigma factors manipulate the geometry of key regions of sigma factors to preventtheir interaction with core-RNAP. Cognate dedicated signal transduction pathways that

release the activities of sigma factors present a dazzling array of mechanismsfromsophisticated protease cascades, through sigma factor mimicry and partner switching, to

the conceptually simple but elegant solution of secretion of an antisigma factor to linkactivity to organelle biogenesis.

3. When free to interact with core-RNAP, all alternative sigma factors must competefiercely with 70 (and each other) for a limited amount of core-RNAP in order to direct

transcription from the promoters they control.

4. Alternative sigma factors are aided in their battle against70 by the alarmone ppGpp

through mechanisms that divert 70

or otherwise counteract its association withcore-RNAP.

FUTURE ISSUES

1. Has the repertoire of mechanisms that can control the availability of alternative sigma

factors reached its limit, or are there future surprises ahead?

2. Do ppGpp and DksA affect transcription mediated by all -RNAP holoenzymes?

3. Does the effect of ppGpp and DksA on sigma factor competition operate purely passively,

or is there an active component involved?

4. How is the dominating 70 subdued to allow alternative sigma factors sufficient access tolimitedcore-RNAP?IsRsdtheonlyanswer,ordoother 70 sequestersexist?DoesRsdact

in concert with a coregulator and/or with Crl-like facilitators of alternative holoenzymeformation?



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DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings that

might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

Apologies are due to all researchers whose original contributions could not be cited due to space

limitations. Our work is supported by the Swedish Research Council (grant number 621-2008-3557 to V.S.) and the European Molecular Biology Organization through a Long Term Research

Fellowship (grant number 540-2009 to T. del P.-S.).

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Annual Review of

Microbiology

Volume 65, 2011 Contents

To the Happy Few

Hiroshi Nikaido

Regulation of DnaA Assembly and Activity: Taking Directions from

the Genome

Alan C. Leonard and Julia E. Grimwade

Regulation of Alternative Sigma Factor Use

Sofia Osterberg, Teresa del Peso-Santos, and Victoria Shingler

Fungal Protein Production: Design and Production

of Chimeric Proteins

Peter J. Punt, Anthony Levasseur, Hans Visser, Jan Wery, and Eric Record

Structure and Function of MARTX Toxins and Other Large

Repetitive RTX Proteins

Karla J.F. Satchell

Eukaryotic Picoplankton in Surface Oceans

Ramon Massana

Life on the Outside: The Rescue ofCoxiella burnetiifrom Its Host CellAnders Omsland and Robert A. Heinzen 1

Molecular Mechanisms ofStaphylococcus aureusIron Acquisition

Neal D. Hammer and Eric P. Skaar 1

Protein Quality Control in the Bacterial Periplasm

Melisa Merdanovic, Tim Clausen, Markus Kaiser, Robert Huber,

and Michael Ehrmann 1

Prospects for the Future Using Genomics and Proteomics

in Clinical Microbiology

Pierre-Edouard Fournier and Didier Raoult

1

The RpoS-Mediated General Stress Response inEscherichia coli

Aurelia Battesti, Nadim Majdalani, and Susan Gottesman 1

Bacterial Osmoregulation: A Paradigm for the Study

of Cellular Homeostasis

Janet M. Wood 2

vi


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Lipoprotein Sorting in Bacteria

Suguru Okuda and Hajime Tokuda 239

Ligand-Binding PAS Domains in a Genomic, Cellular,

and Structural Context

Jonathan T. Henry and Sean Crosson 261

How Viruses and Toxins Disassemble to Enter Host Cells

Takamasa Inoue, Paul Moore, and Billy Tsai

287

Turning Hepatitis C into a Real Virus

Catherine L. Murray and Charles M. Rice 307

Recombination and DNA Repair inHelicobacter pylori

Marion S. Dorer, Tate H. Sessler, and Nina R. Salama 329

Kin Discrimination and Cooperation in Microbes

Joan E. Strassmann, Owen M. Gilbert, and David C. Queller 349

Dinoflagellate Genome Evolution

Jennifer H. Wisecaver and Jeremiah D. Hackett

369Motility and Chemotaxis in CampylobacterandHelicobacter

Paphavee Lertsethtakarn, Karen M. Ottemann, and David R. Hendrixson 389

The Human Gut Microbiome: Ecology and Recent

Evolutionary Changes

Jens Walter and Ruth Ley 411

Approaches to Capturing and Designing Biologically Active Small

Molecules Produced by Uncultured Microbes

Jorn Piel 431

Epidemiological Expansion, Structural Studies, and Clinical

Challenges of New-Lactamases from Gram-Negative Bacteria

Karen Bush and Jed F. Fisher 455

Gene Regulation inBorrelia burgdorferi

D. Scott Samuels 479

Biology ofClostridium difficile:Implications for Epidemiology

and Diagnosis

Karen C. Carroll and John G. Bartlett 501

Interactions of the Human PathogenicBrucellaSpecies

with Their Hosts

Vidya L. Atluri, Mariana N. Xavier, Maarten F. de Jong,

Andreas B. den Hartigh, and Renee M. Tsolis 523

Contents v ii


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Metabolic Pathways Required for the Intracellular Survival

ofLeishmania

Malcolm J. McConville and Thomas Naderer 5

Capsules ofStreptococcus pneumoniaeand Other Bacteria: Paradigms for

Polysaccharide Biosynthesis and Regulation

Janet Yother 5

Synthetic Poliovirus and Other Designer Viruses: What Have WeLearned from Them?

Eckard Wimmer and Aniko V. Paul 5

Regulation of Antigenic Variation inGiardia lamblia

Cesar G. Prucca, Fernando D. Rivero, and Hugo D. Lujan 6

Alternative Pathways of Carbon Dioxide Fixation: Insights into the

Early Evolution of Life?

Georg Fuchs 6

Index

Cumulative Index of Contributing Authors, Volumes 6165 6

Errata

An online log of corrections toAnnual Review of Microbiology articles may be found

http://micro.annualreviews.org/

v ii i Contents


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ANNUAL REVIEWSIts about time. Your time. Its time well spent.

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New From Annual Reviews:

Annual Review of Statistics and Its ApplicationVolume 1 Online January 2014 http://statistics.annualreviews.org

Editor: Stephen E. Fienberg, Carnegie Mellon University

Associate Editors: Nancy Reid, University of Toronto

Stephen M. Stigler, University of Chicago

TheAnnual Review of Statistics and Its Applicationaims to inform statisticians and quantitative methodologi

well as all scientists and users of statistics about major methodological advances and the computational too

allow for their implementation. It will include developments in the eld of statistics, including theoretical stati

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and bioinformatics, economics, machine learning, psychology, sociology, and aspects of the physical scienc

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TABLEOFCONTENTS:

What Is Statistics? Stephen E. Fienberg

A Systematic Statistical Approach to Evaluating Evidence

from Observational Studies, David Madigan, Paul E. Stang,

Jesse A. Berlin, Martijn Schuemie, J. Marc Overhage,

Marc A. Suchard, Bill Dumouchel, Abraham G. Hartzema,

Patrick B. Ryan

The Role of Statistics in the Discovery of a Higgs Boson,

David A. van Dyk

Brain Imaging Analysis, F. DuBois Bowman Statistics and Climate, Peter Guttorp

Climate Simulators and Climate Projections,

Jonathan Rougier, Michael Goldstein

Probabilistic Forecasting, Tilmann Gneiting,

Matthias Katzfuss

Bayesian Computational Tools, Christian P. Robert

Bayesian Computation Via Markov Chain Monte Carlo,

Radu V. Craiu, Jerey S. Rosenthal

Build, Compute, Critique, Repeat: Data Analysis with Latent

Variable Models, David M. Blei

Structured Regularizers for High-Dimensional Problems:Statistical and Computational Issues, Martin J. Wainwright

High-Dimensional Statistics with a View Toward Appli

in Biology, Peter Bhlmann, Markus Kalisch, Lukas M

Next-Generation Statistical Genetics: Modeling, Pena

and Optimization in High-Dimensional Data, Kenneth

Jeanette C. Papp, Janet S. Sinsheimer, Eric M. Sobe

Breaking Bad: Two Decades of Life-Course Data Ana

in Criminology, Developmental Psychology, and Beyo

Elena A. Erosheva, Ross L. Matsueda, Donatello Tele

Event History Analysis, Niels Keiding

Statistical Evaluation of Forensic DNA Prole Evidenc

Christopher D. Steele, David J. Balding

Using League Table Rankings in Public Policy Format

Statistical Issues, Harvey Goldstein

Statistical Ecology, Ruth King

Estimating the Number of Species in Microbial Divers

Studies, John Bunge, Amy Willis, Fiona Walsh

Dynamic Treatment Regimes, Bibhas Chakraborty,

Susan A. Murphy

Statistics and Related Topics in Single-Molecule Biop

Hong Qian, S.C. Kou Statistics and Quantitative Risk Management for Bank

and Insurance, Paul Embrechts, Marius Hofert

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