ReviewArticle - Abertay University · ReviewArticle A Mechanistic Explanation Linking Adaptive Mutation, Niche Change, and Fitness Advantage for the Wrinkly Spreader AndrewJ.Spiers

Review ArticleA Mechanistic Explanation Linking Adaptive Mutation NicheChange and Fitness Advantage for the Wrinkly Spreader

Andrew J Spiers

The SIMBIOS Centre amp School of Science Engineering and Technology Abertay University Bell Street Dundee DD1 1HG UK

Correspondence should be addressed to Andrew J Spiers aspiersabertayacuk

Received 25 June 2013 Accepted 8 November 2013 Published 16 January 2014

Academic Editor Ben-Yang Liao

Copyright copy 2014 Andrew J Spiers This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Experimental evolution studies have investigated adaptive radiation in static liquidmicrocosms using the environmental bacteriumPseudomonas fluorescens SBW25 In evolving populations a novel adaptive mutant known as the Wrinkly Spreader arises withindays having significant fitness advantage over the ancestral strain A molecular investigation of the Wrinkly Spreader has provideda mechanistic explanation linking mutation with fitness improvement through the production of a cellulose-based biofilm atthe air-liquid interface Colonisation of this niche provides greater access to oxygen allowing faster growth than that possiblefor non-biofilmmdashforming competitors located in the lower anoxic region of the microcosm Cellulose is probably normallyused for attachment to plant and soil aggregate surfaces and to provide protection in dehydrating conditions However theevolutionary innovation of the Wrinkly Spreader in static microcosms is the use of cellulose as the matrix of a robust biofilmand is achieved through mutations that deregulate multiple diguanylate cyclases leading to the over-production of cyclic-di-GMPand the stimulation of cellulose expression The mechanistic explanation of the Wrinkly Spreader success is an exemplar of themodern evolutionary synthesis linking molecular biology with evolutionary ecology and provides an insight into the phenomenalability of bacteria to adapt to novel environments

1 Introduction

Competition for limited resources and divergent selectionarising from differences in the environment are key driversof ecological adaptive radiation and ultimately speciation [1]Although usually illustrated by reference to examples suchas Darwinrsquos finches in the Galapagos or the cichlid fishes inEast African Rift Valley lakes [2ndash4] adaptive radiation hasalso played an important role in the great phylogenetic andfunctional diversification of bacteria and can help explain inpart bacterial colonisation and niche preferences as well asbacterial community complexity interactions and dynamics(bacterial adaptive radiation differs in some fundamentalways to that seen in sexual populations [4 5]) Key toadaptive radiation is ecological opportunity which promotesadaptive radiation by changing the selective pressures actingon populations relaxing stabilising selection and creatingconditions that generate diversifying selection [6]The rate atwhich bacterial populations become locally adapted dependson both the selective regime as well as the rate at which adap-tive mutations arise and are fixed within the population In

bacteria adaptations may arise through mutation of existinggenomes and horizontal or lateral gene transfer (HGT)withinpopulations or between phylogenetically similar or distantspecies HGT is generally viewed as the primary means ofacquisition of adaptive mutations for bacteria whereas geneduplications are thought of as the main source of adaptivenovelty in eukaryotes [7 8] Although bacterial adaptive radi-ation can be studied by phylogeographical analyses it is alsoreadily investigated by experimental evolution studies wheregenetic changes and fitness increases have been observed inrelatively short periods (for reviews see [9ndash14])

11 Bacterial Evolution in Simple Microcosms The use ofsimple microcosms for modelling bacterial evolution hasbeen a successful approach because bacterial populations arereadily grown in vitro where they can be initiated with smallisogenic samples have short generation times and reachvery large population sizes During the development of thesepopulations mutations occur randomly and at sufficientrates ensuring that large numbers of novel genotypes appear

Hindawi Publishing CorporationInternational Journal of Evolutionary BiologyVolume 2014 Article ID 675432 10 pageshttpdxdoiorg1011552014675432

2 International Journal of Evolutionary Biology

during the course of the study and are subjected to selectivepressures genetic drift and stochastic events These mutantsare often easily identified by altered phenotypes determinedby growth on agar plates or by simple assays and individualmutants and population samples can be indefinitely stored atminus80∘C allowing comparisons to be made across time pointsand between ancestral and evolved strains In particular thisallows testing of fitness changes where adaptive genotypesare defined as having a competitive fitness (W) advantageover the ancestorW can be calculated as the ratio of Malthu-sian parameters for continuously growing populations whenW gt 1 the mutant has a fitness advantage over the ancestorand is considered an adaptive genotype whenW = 1 the twostrains are neutral with respect to one another and whenWlt 1 the mutant is at a selective disadvantage [15ndash17]

If the possibility of HGT is excluded in experimentalpopulations the origin of all adaptive mutations must bethrough alterations to the ancestral bacterial genome (chro-mosome and accompanying plasmids) andmight range fromsmall-scale sequence changes affecting a single gene to largerrearrangements including deletions or duplications of wholeoperons or considerable portions of the genome These canbe identified through sequencing target genes or throughwhole genome resequencing and the underlying molecularbiology of individual genotypes can be further investigated interms of gene expression patterns regulatory networks andmetabolism in order to understand how adaptive mutationscan be mechanistically linked to altered phenotypes andfitness changes [18]

Although experimental microcosms tend to be physi-cally and chemically simple they can be manipulated tochange ecological opportunities competition environmentalconditions and selective pressures For example glass vialscontaining liquid growth medium can be incubated withconstant shaking to provide a homogeneous environmentor statically where spatial structure becomes important withthemicrocosm becoming heterogeneous and containing newniches at the air-liquid interface the liquid column and vialbottom [19] Nutrient type abundance and complexity aswell as the chemical environment (eg osmolarity O

2 and

pH) can all be manipulated through changes to the growthmediumThe small size low cost and reproducibility of suchmicrocosms mean that they can be used in large numberswith appropriate levels of replication allowing multifactorialexperimental design and including the ability to repeatexperiments using exactly the same initial conditions whenrequired

12 Some Molecular Aspects Underlying Bacterial Evolu-tion In comparison with highly specialized bacteria suchas pathogens and symbionts generalists have a high pro-portion of sensory and regulatory systems which allowthem to respond to a wide range of environmental fac-tors and opportunities These presumably arose throughdistant gene duplication and acquisition events and ofteninvolve multifunctional proteins linking sensory and signaltransduction domains which retain little homology beyondthe functional domains themselves While some regulatory

elements act to modify cellular response or homeostasisvia transcription others modify activity through secondarysignal molecules such as cyclic-di-GMP (bis-(31015840-51015840)-cyclicdimeric guanosine monophosphate) Cyclic-di-GMP is anintracellular signalling molecule that plays a central role inthe regulation of motility virulence and biofilm formationin many bacteria (for reviews see [20ndash24]) It is synthe-sised from GTP (guanosine-51015840-triphosphate) by DGCs (di-guanylate cyclases) and hydrolysed by specific PDEs (phos-phodiesterases) to guanosine monophosphate (GMP) or 51015840-pGpG (linearized di-GMP)which is subsequently hydrolysedto GMP by other hydrolases DGCs are characterised by theamino acid motif Gly-Gly-Asp-Glu-Phe referred to as theGGDEFdomain whilst PDEs include theGlu-Ala-Leu (EAL)or His-Asp-x-Gly-Tyr-Pro (HD-GYP) domains

Bacterial genomes tend to have multiple DGCs andPDEs which suggests that cyclic-di-GMP levels are regulatedthrough a complex signaling network integrating numerousenvironmental signals that control riboswitches transcrip-tion factors and enzyme activities including cellulose expres-sion The disruption of homeostasis through mutation of ahigh-level sensory-regulator systems can have a significantimpact on bacterial phenotype allowing significant fitnessleaps to occur rather than the expected smaller incrementalsteps Furthermore the duplication and divergence of reg-ulators can lead to significant reprogramming of regulatorynetworks [25] with transferred genes (xenologues) generallypersisting in genomes longer than duplicated genes (par-alogues) which tend to have more protein-protein interac-tions and regulators [8]

2 Adaptive Radiation of P fluorescens inStatic Microcosms

The soil and plant-associated fluorescent pseudomonadPseudomonas fluorescens SBW25 has been used as a modelbacterium in experimental evolution studies using simplemicrocosms [19] SBW25 was originally isolated from the leafof a sugar beet (Beta vulgaris) plant [26] and is capable ofcolonising a wide variety of crop plants and weeds Like otherfluorescent pseudomonads it is regarded as a benign plantgrowth-promoting rhizobacterium (ie one that grows onor around the roots of plants in the rhizosphere) HoweverSBW25 carries a defective pathogen-like type III secretionsystem [27] and expresses the virulence-associated cycliclipopeptide class surfactant viscosin [28]Many P fluorescensstrains including SBW25 produce soft rot-like symptomswhen colonising plant tissues following physical damagesuggesting that these soil and plant-associated bacteria arehighly competent colonists with opportunistic pathogenictendencies The ability to colonise new environments is acharacteristic associated with the pseudomonads and maybe enabled by relatively large genomes that include manysensory and regulatory elements [29ndash31] SBW25 sequenceswere initiallymaintained in a partial genomic database beforethe whole genome sequence was determined [30 32] andcomprehensive molecular analyses of regulatory pathwaysmetabolism and fitness are possible using techniques estab-lished for other pseudomonads In addition to experimental

International Journal of Evolutionary Biology 3

evolution studies SBW25 has been used to study plant-microbe interactions (eg [30 33 34]) air-liquid (A-L)interface biofilms and cellulose expression (eg [35ndash39])

In experimental evolution studies SBW25 populationshave been maintained in small glass vials containing KingrsquosB liquid growth medium [40] which is incubated staticallyor with shaking (these are 30ml universal vials containing6ml medium see Figure 1) The adaptive radiation of SBW25in static microcosms is highly reproducible with populationsdiversifying over 3ndash5 days to produce a range of phenotyp-ically distinguishable genotypes occupying different niches[19] (it is notable that in significantly smaller microcosmsSBW25 diversification is far less reproducible suggesting thatthe reproducibility is due in part to larger population sizesand numbers of mutants produced in the larger microcosms[19]) The main genotypes are the Smooth morphs (anabbreviation of morphotypes) which produce round smoothcolonies on Kingrsquos B agar plates and colonise the liquidcolumn of Kingrsquos B medium within static microcosms (theseinclude the ancestral or wild-type SBW25) the Fuzzy Spread-ers which produce stippled colonies and appear to colonisethe bottom of static microcosms and the Wrinkly Spreaderswhich produce a wrinkled colony morphology and colonisethe A-L interface or surface of the liquid column throughthe formation of a visually obvious and robust biofilm (seeFigure 1) (the Wrinkly Spreader phenotype is also observedusing other growth media such Luria Bertani and minimalglucose media) These main genotypes are also referred toas ecomorphs to reflect their niche specialisations withinthe static microcosm As SBW25 reproduction is entirelyasexual in static microcosms (SBW25 does not carry a self-transmissible plasmid nor any other mobile genetic element)these genotypes are analogous to species [41] Variation alsooccurs within genotypes and each can further diversifyto produce the other genotypes though at reduced levelscompared to ancestral SBW25 [42]

A simplistic explanation of the diversification and adap-tation of SBW25 in static microcosms is provided by theRed Queen hypothesis which demands constant evolutionin response to ever-changing competitors and environments[43 44] Competitive interactions between the SmoothFuzzy Spreader and Wrinkly Spreaders result in the stablemaintenance of diversity In most cases populations domi-nated by one genotype can be invaded by a rare genotypewhich can colonise an unoccupied niche and this type ofcompetitive tradeoff between niche specialists is frequencydependent For example the Wrinkly Spreader can invadea population of ancestral SBW25 by colonising the A-Linterface (W = 146) whilst the ancestral SBW25 can invadea population of Wrinkly Spreaders by colonising the liquidcolumn (W = 166) (in five of the six pairwise combinationsthe rare genotype can invade the common one however theFuzzy Spreader is unable to invade the Wrinkly Spreader)[19] Furthermore fitness differences between independentlyisolated Wrinkly Spreaders suggest that there can be strongcompetition within the biofilm itself and as the biofilm getsolder later arisingWrinkly Spreaders have greater fitness thanearlier isolates even though the diversity within the genotypedecreases [45ndash48]

3 Rise of the Wrinkly Spreaders

In wild-type SBW25 populations developing in Kingrsquos B staticmicrocosms Wrinkly Spreaders will appear through randommutation and can represent up to 30ndash50 of the total pop-ulation after five days when biofilms are usually evident [1949 50]Themutation rate of sim10minus7mutationscellgenerationis not unusually elevated in static microcosms and WrinklySpreaders also appear in shaken microcosms where theymight representsim10of the population after the sameperiod

When individual Wrinkly Spreader isolates recovered byspreading microcosm samples on Kingrsquos B agar plates andselecting single colonies are reintroduced into Kingrsquos B staticmicrocosms they produce detectable biofilms covering theentire A-L interface within twelve hours that continue todevelop over 3ndash5 days often reaching 1ndash15mm in depth andcontaining sim106 cellsmL before breaking and sinking [51]A-L interface biofilms are sometimes referred to as pellicles incontrast to the archetypal L-S (liquid-solid surface) interfacebiofilms investigated using flow cells and confocal laserscanning microscopy [52] though biofilms at the meniscusand A-L interface of static liquids form a continuum ofstructures that link A-L A-L-S (air-liquid-solid surface) andL-S biofilms which can be quantitatively differentiated [1339] Unlike static microcosms containing mixed genotypeSBW25 populations in which biofilm material at the A-Linterface and substantial growth in the liquid column isevident Wrinkly Spreader static microcosms show very littlegrowth in the liquid column below the biofilm which oftenappears clear

The rise of the Wrinkly Spreader in static microcosms isexplained by the fitness advantage (W = 15ndash25) these geno-types have over non-biofilm-forming competitors includingthe ancestral SBW25 (the range of W values reflects differ-ences in the assay conditions and the choice of referencestrain) [19 35 49] The evolutionary innovation of theWrinkly Spreader is the production of a biofilm located at theA-L interface of staticmicrocosms sufficient to withstand thenormal spectrumof physical disturbances (ie vibrations andrandom knocks) which allows better access to O

2diffusing

from the atmosphere into the liquid column As a result theWrinkly Spreader shows a new niche preference compared tothe ancestral SBW25

The SBW25 colonists establish an O2gradient within

three hours defining an O2-rich upper zone of sim200120583m and

a lower O2-depleted anoxic zone of sim16mm in Kingrsquos B static

microcosms which persist for up to five days [50] The O2-

rich conditions of the upper zone support higher rates ofgrowth as SBW25 growth is O

2rather than nutrient-limited

in Kingrsquos B medium increasing the chance that a WrinklySpreader mutant will arise in the developing populationWrinkly Spreader cells that are recruited to the A-L interfacewill grow faster than non-biofilm-forming competitors thatcannot maintain a presence in the O

2-rich zone and quickly

form a biofilm and further repress the growth of competitorsGrowth conditions in the biofilm have a significant impacton the physiology of SBW25 cells as biofilm-isolated cells canbe differentiated from those recovered immediately below thebiofilm by Raman spectral profiling [53]


Figure 1 Adaptive radiation in static microcosms gives rise to the Wrinkly Spreader with a new niche preference Shown on the left is a Kingrsquos Bagar plate incubated for three days at 28∘C spreadwith a sample taken from a diversified P fluorescens SBW25 population where both Smoothmorphs and Wrinkly Spreader colonies are evident (this plate has seven Smooth colonies each with a rounded circumference and a smoothconvex surface with one positioned at the top of the plate all of the rest are Wrinkly Spreader colonies which have irregular multilobedcircumferences and a flattened and wrinkled surface) On the right are two static Kingrsquos B microcosms which were incubated for three daysat 28∘CThe left microcosm was inoculated with the wild-type or ancestral SBW25 which grows throughout the liquid column and the rightmicrocosm with the Wrinkly Spreader which colonises the A-L interface through the formation of a robust biofilm

Although both the ancestral SBW25 and the WrinklySpreader can swim using flagella the successful recruitmentof the Wrinkly Spreader probably is the result of alteredcell surface charge or relative hydrophobicity [54] and oncelocated at the meniscus region close to the vial walls WrinklySpreader cells also show a higher level of attachment than theancestral SBW25 [51 54] In this system SBW25 populationsare altering their environment through niche constructionand these changes feedback to influence the subsequentevolution of the Wrinkly Spreaders through ecoevolutionary(ecological-evolutionary) feedbacks where the time scalesof environmental change and evolution are similar [55 56](see the timeline of events in the diversification of SBW25populations in static microcosms leading to the rise of theWrinkly Spreaders in Figure 2)

The Wrinkly Spreader biofilm probably develops frommicrocolonies attached at the meniscus which grow outacross the A-L interface and once the liquid surface iscovered the biofilm develops further by continued growthat the top surface slowly displacing the lower region of thebiofilm further into the liquid column [50 57] This devel-opment requires the cooperation of the growing WrinklySpreader population and the biofilm is the result of the clonalexpansion of a mutant lineage expressing the primary biofilmmatrix material cellulose rather than the result of quorum-based regulation of extracellular polymeric substance orexopolysaccharide (EPS) expression often required for otherbiofilms [52] This cooperation is explained by Hamiltonrsquosinclusive fitness or kin selection theory which states thatcooperation evolves between genetically related individuals[58] and the view of the biofilm structure as a common goodthat is shared by all members of the community is supportedby the finding that non-biofilm-forming cheaters also appearin Wrinkly Spreader biofilms [59]

Ultimately the fitness advantage of the Wrinkly Spreaderis attributable to better O

2access through the formation of a

biofilm at the A-L interface [50] Although the developmentof the biofilm is the result of the cooperation of many gener-ations of Wrinkly Spreaders it can also be viewed as a selfishtrait (of theWrinkly Spreader lineage) and possibly an exam-ple of ancestorrsquos inhibition as the constant production of EPSpushes later generations of cells upwards towards better O

2

conditions and older generations downwards into the anoxicregion where growth is limited [60 61] In contrast in shakenmicrocosms where no O

2gradients can be established the

Wrinkly Spreader has a lower fitness compared to non-biofilm-forming competitors or the ancestral SBW25 (W =sim03ndash10) [35 49] Furthermore onKingrsquos B agar plates wherethe Wrinkly Spreader is genetically unstable and rapidlygenerates Smooth-like revertants (phenotypically similar tothe biofilm cheaters recovered from static microcosms) theWrinkly Spreader has an even lower fitness (W = 015) [62]

4 A Mechanistic Explanation forthe Wrinkly Spreader

The molecular biology underlying the Wrinkly Spreader(WS) phenotypewas first investigated using amini-Tn5 trans-poson screening approach in order to identify critical genesand regulatory pathways required for a wrinkled colony andA-L interface biofilm formation [35] (the archetypal WrinklySpreader referred to here is a specific strain also recorded asPR1200 [35] and Large Spreading Wrinkly Spreader (LSWS)[46]) Two sets of Wrinkly Spreader mini-Tn5 mutants wererecovered and used to characterise the wsp and wss operonsresponsible for the regulation and production of the WSphenotype respectively [35 46 51 54 63 64]


Timeline

Epoch

Genotypes

and niches

Events and

consequenceslowast

In the first sim3 hours

Environmental change and nicheconstruction

Ancestral SBW25 in the liquidcolumn

Initial colonists alter thehomogeneous environment of the

static microcosm by formingan O2 gradient

Establishing a high O2 niche at the

From the first day

Diversification

Wrinkly Spreaders at the A-Linterface

Smooth morphs in the liquid column

Fuzzy Spreaders at the anoxicbottom

Wrinkly Spreader mutation in a CDGor CDG regulator results in increased

levels of cyclic-di-GMP

Activation of the cellulose synthaseexpression of attachment factor andrecruitment to the A-L interface

Formation of a robust biofilm and theearly interception of O2 diffusing into

the liquid column

up to five or more days

Rise of the Wrinkly Spreaders

Competition and diversification inthe biofilm

(Similarly)

(Similarly)

Wrinkly Spreaders enjoy faster

growth and higher fitness than

non-biofilm-forming competitors

A-L interface and a low O2 nichelower down the liquid column

Figure 2 Linking the timeline of adaptive radiation to events and consequences in the rise of the Wrinkly Spreader The adaptive radiation ofP fluorescens SBW25 in static microcosms can be mapped to a timeline and epochs during which the ancestral strain diversifies into newgenotypes with altered niche preferences The events and consequences of mutation are outlined for the Wrinkly Spreader along the bottompanel of the figure Although not shown here further diversification will occur amongst the Smooth morphs and Fuzzy Spreaders

41 From the wsp Regulatory Operon to the Mutations thatActivate the WS Phenotype The wsp regulatory operonconsists of seven genes (wspA-F and wspR) showing sig-nificant protein level homology with chemosensory signal-transduction-like operons (wsp is an acronym forWS pheno-type reflecting the fact that this operon encodes a regulatorycomponent required for the WS phenotype) [35 46] Thefunctioning of the Wsp system (see Figure 3) has beenmodelled on theChe chemosensory systemofEscherichia coli[65] to provide a mechanistic explanation of the inductionof the WS phenotype [46] Based on the behaviour ofthe homologous Wsp proteins in P aeruginosa PA01 [66]WspA WspB WspC WspD WspE and WspF are likelyto form a membrane-associated receptor-signaling complexthat responds to stimulus associated with the growth on asolid surface [67] by activating the associatedWspR responseregulator (SBW25 wsp-like operons are also found in therelated pseudomonads P fluorescens Pf0-1 P putidaKT2440and P syringae pv tomato DC3000 see the PseudomonasGenome Database [68])

WspR is a DGC which catalyses the synthesis of cyclic-di-GMP when phosphorylated [64] In wild-type SBW25WspR activity is modulated by the opposing activities of theWspC and WspF subunits However wspF mutations thatare predicted to reduce or abolish WspF function have beenidentified in a number of independently isolated WrinklySpreaders [46] Allele-exchange experiments have shown thatawspFmutation is necessary and sufficient for theWSpheno-type converting wild-type SBW25 into a Wrinkly Spreader

Conversely the replacement of a wspF mutation with thewild-type sequence reverts the strain back to the ancestralphenotype [46] SubsequentlywspEmutations have also beenidentified in independently isolated Wrinkly Spreaders [69]In these the activity of WspE may be increased leadingdirectly to the overactivation of WspR Perhaps surprisinglynowspRmutations have been identified in independently iso-latedWrinkly Spreaders to dateHowevermutations affectingtwo other DGCs AwsR andMwsR and a putative DGC-PDEhybrid SwsR are also known to induce the WS phenotype[34 69ndash71] Although three different DGCs appear to betargeted by adaptive mutation it is noteworthy that theSBW25 genome contains thirty-nine putative genes encodingDGCs [30] Similarly in Pf0-1 four of thirty putative DGCsappear to be involved in biofilm formation [72] Systematicanalyses of these show that one DGC preferentially affectsthe localisation of the primary Pf0-1 protein adhesin LapAanother controls swimming and the last affects both LapAand motility These findings suggest that different cyclic-di-GMP-regulated systems can be specifically controlled bydistinct DGCs [72]

42 The Involvement of Cellulose in the WS PhenotypeThe cyclic-di-GMP catalysed by WspR or other DGCs ina number of independently isolated Wrinkly Spreaders isbelieved to activate a membrane-associated cellulose syn-thase complex encoded by the ten gene wss operon (wssA-J) (wss is an acronym for WS structural reflecting the factthat this operon encodes a structural component required


GMP

WspA

WspC WspF

WspDWspB

WspE

WspR

Innermembrane

+CH3 minusCH3

Cyclic-di-GMP

Figure 3 The Wsp system is responsible for the synthesis of cyclic-di-GMP and the activation of the Wrinkly Spreader (WS) phenotypeThe functioning of the P fluorescens SBW25 Wsp system has been modelled on the Che chemosensory system of E coli and providesa mechanistic explanation linking adaptive mutations to the WS phenotype The methyl-accepting chemotaxis protein (WspA) scaffoldproteins (WspB and WspD) and histidine kinase (WspE) form a membrane-associated receptor-signaling complex In the absence of anappropriate environmental signal the complex is silent Upon activation by phosphorylation (indicated by the black circles) the diguanylatecyclase (DGC) response regulator (WspR) synthesizes cyclic-di-GMP from GTP The system is controlled by the opposing activities of amethyltransferase (WspC) and methylesterase (WspF) which add and remove respectively methyl (CH

3

) groups on the signalling domainof WspA (circles) In wild-type SBW25 the activities of the two are balanced preventing the activation of WspR and allowing the Wspcomplex to oscillate between active and inactive states Mutations inhibiting WspF function or activating WspE kinase activity result in theactivation ofWspR and the production of cyclic-di-GMP Increased levels of cyclic-di-GMP then lead to the expression of theWS phenotypeThe Wsp system is shown as a schematic only the three-dimensional structure of the proteins their relative placement numbers and thepositioning of the complex in the inner membrane have not yet been determined

for the WS phenotype) [35] WssB WssC WssD and WssEshow significant protein level homology to the core cellulosesynthase subunits originally identified in the bcs (bacterialcellulose synthesizing) operons of Acetobacter xylinum (nowknown as Gluconacetobacter hansenii ATCC 23769 [73])and E coli K-12 [74] with WssB identified as the cyclic-di-GMP-binding catalytically active subunit responsible for thepolymerisation of UDP glucose into cellulose [35] Howeverthe wss operon contains additional genes (wssA and wssF-J) not previously recognised as having a role in cellulosesynthesis WssA and WssJ are MinD-like homologues andmay be responsible for the correct spatial localization of theWss cellulose synthase complex at the cell poles as is the casefor the K12 YhjQ-BcsQ WssA homologue [75] and WssF ispredicted to provide acyl groups to WssG WssH and WssIwhich share homology with the AlgF AlgI and AlgJ alginateacetylation proteins of P aeruginosa FRD1 [76]

Although bcs operons are widespread amongst bacteria[39] only DC3000 has a complete wss operon includingthe acetylation-associated genes wssF-I whilst KT2440 hasa truncated wssA-E operon (see the Pseudomonas GenomeDatabase [68]) and both have been shown to express cellu-lose experimentally [36] (in contrast PA01 and Pf0-1 do not

contain bcs operons and utilise other matrix components intheir biofilms [77 78])

The expression of extracellular cellulose by the WrinklySpreader was confirmed by comparative Congo red stainingof colonies Calcofluor-based fluorescent microscopy andcellulase digestion of biofilm material and was chemicallyidentified as partially acetylated cellulose by the structuralanalysis of purified biofilmmatrix material [35 51] (reviewedby [39]) Significantly a Wrinkly Spreader wspR mini-Tn5mutant does not express cellulose whilst the addition of aplasmid-borne constitutively active WspR mutant in wild-type SBW25 expresses cellulose and produces theWS pheno-type [35 51 54 63] Further minitransposon analysis of theWrinkly Spreader has confirmed that all of the wsp and wssgenes except wssJ are required for the WS phenotype andthat WssJ may be functionally redundant [70]

TheWrinkly Spreader biofilmmatrix appears as an exten-sive network of extracellular cellulose with 002ndash100120583mthick fibres forming thin films around voids and linking largeclumps ofmaterial [51]The structure itself is highly hydratedcontaining 97 liquid and having a density almost equivalentto that of the culturemediaWithin the biofilm bacterial cellsare associated with the cellulose fibres and are also found


within the voids [51] Scanning electron microscopy andconfocal laser scanning microscopy suggest that the biofilmmay be a lattice work of pores produced by constant growthat the top surface of the biofilm which slowly displaces olderstrata deeper into the liquid column [39 50] The degreeof wrinkleality (or wrinkledness) [13] of Wrinkly Spreaderbiofilms and colonies depends on interactions between thecellulose fibres and cells as well as lipopolysaccharide andan unidentified fimbriae-like attachment factor also overex-pressed by the Wrinkly Spreader [54] Wrinkleality can bequantified using a combination of assays including colonyexpansion reversion rates growth in static microcosmsbiofilmattachment levels and strength [38 51 62] andused todifferentiate betweenWrinkly Spreader isolates (unpublishedobservations A Spiers amp Y Udall)

43 The Wrinkly Spreader Is Not the Only Genotype Capableof Exploiting the A-L Interface In addition to the WrinklySpreader SBW25 has been observed to produce two addi-tional A-L interface biofilms When induced nonspecificallywith iron (FeCl

3) wild-type SBW25 will produce a fragile

cellulose-based viscous mass (VM) biofilm which is poorlyattached at the meniscus and substantially weaker than theWrinkly Spreader biofilm [37] This physiologically inducedbiofilm is phenotypically indistinguishable from that pro-duced by JB01 a SBW25 strain in which the neomycin phos-photransferase gene promoter (nptII) was inserted upstreamof the wss operon to increase transcription and celluloseexpression [35] (wild-type SBW25 expresses low levels ofcellulose when grown in Kingrsquos B medium suggesting thatthere is some cyclic-di-GMP available to induce the cellulosesynthase complex [35 51]) This suggests that mutants thatproduce VM-like biofilms are likely to appear in diversifyingpopulations of wild-type SBW25 perhaps increasing wsspromoter activity directly or indirectly by reducing thefunctioning of wss transcriptional repressors It is possiblethat such mutants have escaped attention as they would beexpected to produce Smooth-like colonies similar to thoseproduced by JB01

Complementary biofilm-forming strain (CBFS) mutantsof a cellulose-deficient SBW25 strain have also been isolatedfrom static microcosms though for these the mechanismunderlying biofilm-formation is as yet unknown [70] Acomparison of all three biofilm types using a common non-biofilm-forming reference strain indicates that all provide afitness advantage in static microcosms (W = 27 22 and 18for CBFS VM andWS resp) and can be differentiated on thebasis of a number of quantitative biofilm-associated assayssuggesting that they represent three different solutions to thecolonisation of the A-L interface (unpublished observationsA Spiers amp A Koza) It is not yet clear why the WrinklySpreader appears to be the most successful biofilm typearising in diversifying populations of SBW25 However itis possible that the biofilms produced by CBFS or VM-likemutants may be more costly physically unreliable or struc-turally overengineered compared to the Wrinkly SpreaderAlternatively the genetic architecture of SBW25 may favourmutations that result in the WS phenotype rather than apartial VM phenotype where only cellulose expression is

activated or the activation of an entirely different pathwayleading to the CBFS phenotype

5 Possible Role of Cellulose inNatural Environments

Although the ability to produce cellulose-based A-L interfacebiofilms in static microcosms is common amongst environ-mental pseudomonads [36 38 79] (reviewed by [39]) it isunclear what the functional role of cellulose might be in thenatural habitats of these bacteria Whilst biofilm-formationis a key bacterial strategy for colonisation it is only oneof a range of assemblages that bacteria can form rangingfrom isolated surface-attached bacteria microcolonies andmultilayeredmultispecies and differentiated biofilms to flocsand slime (for reviews see [52 78 80ndash84]) In contrast tothe view of biofilm formation as a genetically determineddevelopmental programme [85] biofilms are transient com-munities better described by adaptation social evolution andecological succession [58 81 86] In these the Red Queenmay drive competition and adaptation in nascent biofilmsbut the Black Queen may play a greater role in developinglonger lasting and more robust interdependent cooperativecommunities in older more permanent structures [87 88]

It is possible that biofilm-formation is used by envi-ronmental pseudomonads to rapidly colonise the meniscusand A-L interface of temporary water bodies such as thosefound in partially saturated soil pore networks or collectedon surfaces after rainfall However the paradigm of theimmersed cellulose matrix-based biofilm exemplified by theWrinkly Spreader is challenged by the finding that celluloseexpression by SBW25 provides a fitness advantage in naturalenvironments where water does not collect or remain forany length of time Competitive fitness assays have shownthat wild-type SBW25 has a fitness advantage comparedto a cellulose-deficient mutant of W = 18 on the leavesof sugar beet seedlings and W = 11 on the roots [33]Recent experimentation has shown that SBW25 colonizesmushroom (Agaricus bisporus) caps where it can produceblotch-like disease symptoms similar to other Pseudomonasspp mushroom pathogens and in these situations the abilityto express cellulose also provides a fitness advantage ofW = 12ndash14 (unpublished observations A Spiers amp AKoza) An alternative role for cellulose in these naturalnonsaturated environments is suggested by the finding thatcellulose expression also enhances the survival of SBW25under dehydrating or low humidity conditions (unpublishedobservations A Spiers amp A Koza) where cellulose fibresmight help to retain water around microcolonies and trapwater vapour directly from the air [82 89] Conceivably thecellulose matrix may also help with nutrient acquisition (egfrom root exudates and soil aggregates) and retention

6 Concluding Statement

Even in simple microcosms ecological opportunity andcompetition between genotypes can act to drive the adaptiveradiation of bacterial populations In the case of SBW25 it


appears that the presence of multiple DGCs in the genomeresulting from gene duplications and acquisitions deep in thephylogenetic history of the strain predisposes it to adaptivemutation niche change and fitness leaps in static liquidmicrocosms leading to the rise of the Wrinkly SpreadersThe molecular biology underlying the WS phenotype is nowwell understood providing amechanistic explanation linkingadaptive mutations which activate DGCs to overproducecyclic-di-GMP the expression of cellulose and the formationof biofilms at the A-L interface with the fitness benefitobtained by the colonisation of this new niche over non-biofilm-forming competitors and the ancestral SBW25 strain

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This paper was written with support from the SIMBIOSCentre at the Abertay University A Spiers is also memberof the Scottish Alliance for Geoscience Environment andSociety (SAGES) The unpublished work cited here wasundertaken by Yvette Udall and Anna Koza during theirMasters andDoctoral studies with A Spiers respectivelyTheAbertay University is a charity registered in Scotland noSC016040

References

[1] D SchluterThe Ecology of Adaptive Radiation Oxford Series inEcology and Evolution Oxford University Press Oxford UK1st edition 2000

[2] D Lack Darwinrsquos Finches Cambridge University Press Cam-bridge UK 1947

[3] G Fryer and T D Iles The Cichlid Fishes of the Great Lakes ofAfrica Oliver and Boyd Edinburgh UK 1972

[4] MHau andMWikelski ldquoDarwinrsquos Finchesrdquo inELS JohnWileyamp Sons Chichester UK 2001

[5] B R Levin and C T Bergstrom ldquoBacteria are different obser-vations interpretations speculations and opinions about themechanisms of adaptive evolution in prokaryotesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 97 no 13 pp 6981ndash6985 2000

[6] J B Yoder E Clancey S Des Roches et al ldquoEcologicalopportunity and the origin of adaptive radiationsrdquo Journal ofEvolutionary Biology vol 23 no 8 pp 1581ndash1596 2010

[7] R J Whitaker ldquoAllopatric origins of microbial speciesrdquo Philo-sophical Transactions of the Royal Society B vol 361 no 1475pp 1975ndash1984 2006

[8] T J Treangen and E P C Rocha ldquoHorizontal transfer notduplication drives the expansion of protein families in prokary-otesrdquo PLoS Genetics vol 7 no 1 Article ID article e10012842011

[9] P B Rainey A Buckling R Kassen and M Travisano ldquoTheemergence and maintenance of diversity insights from experi-mental bacterial populationsrdquo Trends in Ecology and Evolutionvol 15 no 6 pp 243ndash247 2000

[10] J Adams ldquoMicrobial evolution in laboratory environmentsrdquoResearch in Microbiology vol 155 no 5 pp 311ndash318 2004

[11] R C MacLean ldquoAdaptive radiation in microbial microcosmsrdquoJournal of Evolutionary Biology vol 18 no 6 pp 1376ndash13862005

[12] A Buckling R C MacLean M A Brockhurst and N Cole-grave ldquoThe beagle in a bottlerdquo Nature vol 457 no 7231 pp824ndash829 2009

[13] A J Spiers ldquoBacterial evolution in simple microcosmsrdquo inMicrocosms Ecology Biological Implications and EnvironmentalImpact C H Harris Ed Microbiology Research AdvancesSeries Nova Publishers Hauppauge NY USA 2013

[14] J NThompson Relentless Evolution The University of ChicagoPress Chicago Ill USA 2013

[15] R E Lenski M R Rose S C Simpson and S C Tadler ldquoLong-term experimental evolution in Escherichia coli I adaptationand divergence during 2000 generationsrdquo The American Natu-ralist vol 138 no 6 pp 1315ndash1341 1991

[16] H A Orr ldquoFitness and its role in evolutionary geneticsrdquoNatureReviews Genetics vol 10 no 8 pp 531ndash539 2009

[17] L-M Chevin ldquoOn measuring selection in experimental evolu-tionrdquo Biology Letters vol 7 no 2 pp 210ndash213 2011

[18] A C Dalziel S M Rogers and P M Schulte ldquoLinkinggenotypes to phenotypes and fitness how mechanistic biologycan inform molecular ecologyrdquo Molecular Ecology vol 18 no24 pp 4997ndash5017 2009

[19] P B Rainey andMTravisano ldquoAdaptive radiation in a heteroge-neous environmentrdquoNature vol 394 no 6688 pp 69ndash72 1998

[20] R Hengge ldquoPrinciples of c-di-GMP signalling in bacteriardquoNature Reviews Microbiology vol 7 no 4 pp 263ndash273 2009

[21] T L Povolotsky andRHengge ldquolsquoLife-style rsquo control networks inEscherichia coli signaling by the second messenger c-di-GMPrdquoJournal of Biotechnology vol 160 no 1-2 pp 10ndash16 2012

[22] D Srivastava and C M Waters ldquoA tangled web regulatoryconnections between quorum sensing and cyclic di-GMPrdquoJournal of Bacteriology vol 194 no 17 pp 4485ndash4493 2012

[23] H Sondermann N J Shikuma and F H Yildiz ldquoYoursquovecome a long way c-di-GMP signalingrdquo Current Opinion inMicrobiology vol 15 no 2 pp 140ndash146 2012

[24] U RomlingM Y Galperin andMGomelsky ldquoCyclic di-GMPthe first 25 years of a universal bacterial second messengerrdquoMicrobiology and Molecular Biology Reviews vol 77 no 1 pp1ndash52 2013

[25] L Wang F F Wang and W Qian ldquoEvolutionary rewiring andreprogramming of bacterial transcription regulationrdquo Journal ofGenetics and Genomics vol 38 no 7 pp 279ndash288 2011

[26] P B Rainey and M J Bailey ldquoPhysical and genetic map ofthe Pseudomonas fluorescens SBW25 chromosomerdquo MolecularMicrobiology vol 19 no 3 pp 521ndash533 1996

[27] GM Preston N Bertrand and P B Rainey ldquoType III secretionin plant growth-promoting Pseudomonas fluorescens SBW25rdquoMolecular Microbiology vol 41 no 5 pp 999ndash1014 2001

[28] I de Bruijn M J D de Kock M Yang P de Waard T A VanBeek and J M Raaijmakers ldquoGenome-based discovery struc-ture prediction and functional analysis of cyclic lipopeptideantibiotics in Pseudomonas speciesrdquo Molecular Microbiologyvol 63 no 2 pp 417ndash428 2007

[29] A J Spiers A Buckling and P B Rainey ldquoThe causes ofPseudomonas diversityrdquoMicrobiology vol 146 no 10 pp 2345ndash2350 2000


[30] M W Silby A M Cerdeno-Tarraga G S Vernikos et alldquoGenomic and genetic analyses of diversity and plant interac-tions of Pseudomonas fluorescensrdquo Genome Biology vol 10 no5 article R51 2009

[31] M W Silby C Winstanley S A C Godfrey S B Levy andR W Jackson ldquoPseudomonas genomes diverse and adaptablerdquoFEMS Microbiology Reviews vol 35 no 4 pp 652ndash680 2011

[32] A J Spiers D Field M Bailey and P B Rainey ldquoNoteson designing a partial genomic database the Pf SBW25 ency-clopaedia a sequence database for Pseudomonas fluorescensSBW25rdquoMicrobiology vol 147 no 2 pp 247ndash249 2001

[33] MGalGM Preston RCMasseyA J Spiers andP B RaineyldquoGenes encoding a cellulosic polymer contribute toward theecological success of Pseudomonas fluorescens SBW25 on plantsurfacesrdquoMolecular Ecology vol 12 no 11 pp 3109ndash3121 2003

[34] S RGiddens RW Jackson CDMoon et al ldquoMutational acti-vation of niche-specific genes provides insight into regulatorynetworks and bacterial function in a complex environmentrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 104 no 46 pp 18247ndash18252 2007

[35] A J Spiers S G Kahn J Bohannon M Travisano and P BRainey ldquoAdaptive divergence in experimental populations ofPseudomonas fluorescens I Genetic and phenotypic bases ofwrinkly spreader fitnessrdquo Genetics vol 161 no 1 pp 33ndash462002

[36] S Ude D L Arnold C D Moon T Timms-Wilson and AJ Spiers ldquoBiofilm formation and cellulose expression amongdiverse environmental Pseudomonas isolatesrdquo EnvironmentalMicrobiology vol 8 no 11 pp 1997ndash2011 2006

[37] A Koza P D Hallett C D Moon and A J Spiers ldquoCharacter-ization of a novel air-liquid interface biofilm of Pseudomonasfluorescens SBW25rdquoMicrobiology vol 155 no 5 pp 1397ndash14062009

[38] M Robertson S M Hapca O Moshynets and A J SpiersldquoAir-liquid interface biofilm formation by psychrotrophic pseu-domonads recovered from spoilt meatrdquo Antonie Van Leeuwen-hoek vol 103 no 1 pp 251ndash259 2013

[39] A J Spiers Y Y Deeni AO Folorunso A Koza OMoshynetsand K Zawadzki ldquoCellulose expression in Pseudomonas flu-orescens SBW25 and other environmental pseudomonadsrdquo inCellulose T G M Van De Ven and L Godbout Eds InTechRijeka Croatia 2013

[40] E O King M K Ward and D E Raney ldquoTwo simple mediafor the demonstration of pyocyanin and fluorescinrdquoThe Journalof Laboratory and Clinical Medicine vol 44 no 2 pp 301ndash3071954

[41] R Kassen A Buckling G Bell and P B Ralney ldquoDiversitypeaks at intermediate productivity in a laboratory microcosmrdquoNature vol 406 no 6795 pp 508ndash512 2000

[42] A Buckling M A Wills and N Colegrave ldquoAdaptation limitsdiversification of experimental bacterial populationsrdquo Sciencevol 302 no 5653 pp 2107ndash2109 2003

[43] L Van Valen ldquoA new evolutionary lawrdquo Evolutionary Theoryvol 1 pp 1ndash30 1973

[44] L H Liow L Van Valen and N C Stenseth ldquoRed queen frompopulations to taxa and communitiesrdquo Trends in Ecology andEvolution vol 26 no 7 pp 349ndash358 2011

[45] M A Brockhurst M E Hochberg T Bell and A BucklingldquoCharacter displacement promotes cooperation in bacterialbiofilmsrdquo Current Biology vol 16 no 20 pp 2030ndash2034 2006

[46] E Bantinaki R Kassen C G Knight Z Robinson A JSpiers and P B Rainey ldquoAdaptive divergence in experimentalpopulations of Pseudomonas fluorescens III Mutational originsof wrinkly spreader diversityrdquo Genetics vol 176 no 1 pp 441ndash453 2007

[47] D J P Engelmoer and D E Rozen ldquoFitness trade-offs mod-ify community composition under contrasting disturbanceregimes in pseudomonas fluorescens microcosmsrdquo Evolutionvol 63 no 11 pp 3031ndash3037 2009

[48] J R Meyer S E Schoustra J Lachapelle and R Kassen ldquoOver-shooting dynamics in a model adaptive radiationrdquo Proceedingsof the Royal Society B vol 278 no 1704 pp 392ndash398 2011

[49] J H Green A Koza O Moshynets R Pajor M R Ritchieand A J Spiers ldquoEvolution in a test tube rise of the wrinklyspreadersrdquo Journal of Biological Education vol 45 no 1 pp 54ndash59 2011

[50] A Koza O Moshynets W Otten and A J Spiers ldquoEnvi-ronmental modification and niche construction developingO2

gradients drive the evolution of the wrinkly spreaderrdquoInternational Society of Microbial Ecology Journal vol 5 no 4pp 665ndash673 2011

[51] A J Spiers J Bohannon SMGehrig andP B Rainey ldquoBiofilmformation at the air-liquid interface by the Pseudomonas fluo-rescens SBW25 wrinkly spreader requires an acetylated form ofcelluloserdquoMolecularMicrobiology vol 50 no 1 pp 15ndash27 2003

[52] S S Branda A Vik L Friedman and R Kolter ldquoBiofilms thematrix revisitedrdquo Trends in Microbiology vol 13 no 1 pp 20ndash26 2005

[53] W E Huang S Ude and A J Spiers ldquoPseudomonas fluorescensSBW25 biofilm and planktonic cells have differentiable Ramanspectral profilesrdquo Microbial Ecology vol 53 no 3 pp 471ndash4742007

[54] A J Spiers and P B Rainey ldquoThe Pdeudomonas fluorescensSBW25 wrinkly spreader biofilm requires attachment factorcellulose fibre and LPS interactions to maintain strength andintegrityrdquoMicrobiology vol 151 no 9 pp 2829ndash2839 2005

[55] F Pelletier D Garant and A P Hendry ldquoEco-evolutionarydynamicsrdquo Philosophical Transactions of the Royal Society B vol364 no 1523 pp 1483ndash1489 2009

[56] D M Post and E P Palkovacs ldquoEco-evolutionary feedbacksin community and ecosystem ecology interactions betweenthe ecological theatre and the evolutionary playrdquo PhilosophicalTransactions of the Royal Society B vol 364 no 1523 pp 1629ndash1640 2009

[57] O V Moshynets A Koza P Dello Sterpaio V A Kordium andA J Spiers ldquoUp-dating the Cholodny method using PET filmsto sample microbial communities in soilrdquo Biopolymers and Cellvol 27 no 3 pp 199ndash205 2011

[58] S A West A S Griffin A Gardner and S P Diggle ldquoSocialevolution theory for microorganismsrdquoNature Reviews Microbi-ology vol 4 no 8 pp 597ndash607 2006

[59] Q G Zhang A Buckling R J Ellis and H C J GodfrayldquoCoevolution between cooperators and cheats in a microbialsystemrdquo Evolution vol 63 no 9 pp 2248ndash2256 2009

[60] J B Xavier and K R Foster ldquoCooperation and conflict inmicrobial biofilmsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 104 no 3 pp 876ndash881 2007

[61] F Baquero and M Lemonnier ldquoGenerational coexistence andancestorrsquos inhibition in bacterial populationsrdquo FEMSMicrobiol-ogy Reviews vol 33 no 5 pp 958ndash967 2009


[62] A J Spiers ldquoWrinkly-spreader fitness in the two-dimensionalagar plate microcosm maladaptation compensation and eco-logical successrdquo PLoS One vol 2 no 1 article e740 2007

[63] P Goymer S G Kahn J G Malone S M Gehrig A JSpiers and P B Rainey ldquoAdaptive divergence in experimentalpopulations of Pseudomonas fluorescens II Role of the GGDEFregulator WspR in evolution and development of the wrinklyspreader phenotyperdquoGenetics vol 173 no 2 pp 515ndash526 2006

[64] J G Malone R Williams M Christen U Jenal A J Spiersand P B Rainey ldquoThe structure-function relationship ofWspRa Pseudomonas fluorescens response regulator with a GGDEFoutput domainrdquoMicrobiology vol 153 no 4 pp 980ndash994 2007

[65] A Bren and M Eisenbach ldquoHow signals are heard duringbacterial chemotaxis protein-protein interactions in sensorysignal propagationrdquo Journal of Bacteriology vol 182 no 24 pp6865ndash6873 2000

[66] J W Hickman D F Tifrea and C S Harwood ldquoA chemosen-sory system that regulates biofilm formation through modu-lation of cyclic diguanylate levelsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no40 pp 14422ndash14427 2005

[67] Z T Guvener and C S Harwood ldquoSubcellular location charac-teristics of thePseudomonas aeruginosaGGDEFproteinWspRindicate that it produces cyclic-di-GMP in response to growthon surfacesrdquo Molecular Microbiology vol 66 no 6 pp 1459ndash1473 2007

[68] G L Winsor D K W Lam L Fleming et al ldquoPseudomonasgenome database improved comparative analysis and popula-tion genomics capability for Pseudomonas genomesrdquo NucleicAcids Research vol 39 no 1 pp D596ndashD600 2011

[69] M J McDonald S M Gehrig P L Meintjes X X Zhang andP B Rainey ldquoAdaptive divergence in experimental populationsof Pseudomonas fluorescens IV Genetic constraints guide evo-lutionary trajectories in a parallel adaptive radiationrdquo Geneticsvol 183 no 3 pp 1041ndash1053 2009

[70] S M Gehrig Adaptation of Pseudomonas fluorescens SBW25 tothe air-liquid interface a study in evolutionary genetics [PhDthesis] University of Oxford Oxford UK 2005

[71] M J McDonald T F Cooper H J E Beaumont and P BRainey ldquoThe distribution of fitness effects of new beneficialmutations in Pseudomonas fluorescensrdquo Biology Letters vol 7no 1 pp 98ndash100 2011

[72] P D Newell S Yoshioka K L Hvorecny R D Monds andG A OrsquoToole ldquoSystematic analysis of diguanylate cyclases thatpromote biofilm formation by Pseudomonas fluorescens Pf0-1rdquoJournal of Bacteriology vol 193 no 18 pp 4685ndash4698 2011

[73] I M Saxena K Kudlicka K Okuda and R M Brown JrldquoCharacterization of genes in the cellulose-synthesizing operon(acs operon) of Acetobacter xylinum implications for cellulosecrystallizationrdquo Journal of Bacteriology vol 176 no 18 pp 5735ndash5752 1994

[74] F R Blattner G Plunkett III C A Bloch et al ldquoThe completegenome sequence of Escherichia coli K-12rdquo Science vol 277 no5331 pp 1453ndash1462 1997

[75] B Le Quere and J M Ghigo ldquoBcsQ is an essential componentof the Escherichia coli cellulose biosynthesis apparatus thatlocalizes at the bacterial cell polerdquoMolecular Microbiology vol72 no 3 pp 724ndash740 2009

[76] M J Franklin and D E Ohman ldquoIdentification of algI andalgI in the Pseudomonas aeruginosa alginate biosynthetic genecluster which are required for alginate O acetylationrdquo Journal ofBacteriology vol 178 no 8 pp 2186ndash2195 1996

[77] R D Monds P D Newell R H Gross and G A OrsquoTooleldquoPhosphate-dependent modulation of c-di-GMP levels regu-lates Pseudomonas fluorescens Pf0-1 biofilm formation by con-trolling secretion of the adhesin LapArdquoMolecularMicrobiologyvol 63 no 3 pp 656ndash679 2007

[78] D Lopez H Vlamakis and R Kolter ldquoBiofilmsrdquo Cold SpringHarbor Perspectives in Biology vol 2 no 7 article a000398 2010

[79] A J Spiers D L Arnold C DMoon and TM Timms-WilsonldquoA survey of A-L biofilm formation and cellulose expressionamongst soil and plant-associated Pseudomonas isolatesrdquo inMicrobial Ecology of Aerial Plant Surfaces M J Bailey A KLilley T M Timms-Wilson and P T N Spencer-Phillips Edspp 121ndash132 CABI Wallingford UK 2006

[80] L Hall-Stoodley J W Costerton and P Stoodley ldquoBacterialbiofilms from the natural environment to infectious diseasesrdquoNature Reviews Microbiology vol 2 no 2 pp 95ndash108 2004

[81] T J Battin W T Sloan S Kjelleberg et al ldquoMicrobiallandscapes new paths to biofilm researchrdquo Nature ReviewsMicrobiology vol 5 no 1 pp 76ndash81 2007

[82] H C Flemming and JWingender ldquoThe biofilmmatrixrdquoNatureReviews Microbiology vol 8 no 9 pp 623ndash633 2010

[83] S Elias and E Banin ldquoMulti-species biofilms living withfriendly neighborsrdquo FEMS Microbiology Reviews vol 36 no 5pp 990ndash1004 2012

[84] O Rendueles and J M Ghigo ldquoMulti-species biofilms how toavoid unfriendly neighborsrdquo FEMS Microbiology Reviews vol36 no 5 pp 972ndash989 2012

[85] G OrsquoToole H B Kaplan and R Kolter ldquoBiofilm formation asmicrobial developmentrdquoAnnual Review ofMicrobiology vol 54pp 49ndash79 2000

[86] J-U Kreft and S Bonhoeffer ldquoThe evolution of groups ofcooperating bacteria and the growth rate versus yield trade-offrdquoMicrobiology vol 151 no 3 pp 637ndash641 2005

[87] J J Morris R E Lenski and E R Zinser ldquoThe black queenhypothesis evolution of dependencies through adaptive genelossrdquoMBio vol 3 no 2 article e00036-12 2012

[88] J L Sachs and A C Hollowell ldquoThe origins of cooperativebacterial communitiesrdquo MBio vol 3 no 3 article e00099-122012

[89] E Hoshino Y Wada and K Nishizawa ldquoImprovements in thehygroscopic properties of cotton cellulose by treatment with anendo-type cellulase from Streptomyces sp KSM-26rdquo Journal ofBioscience and Bioengineering vol 88 no 5 pp 519ndash525 1999

Impact Factor 173028 Days Fast Track Peer ReviewAll Subject Areas of ScienceSubmit at httpwwwtswjcom

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal


during the course of the study and are subjected to selectivepressures genetic drift and stochastic events These mutantsare often easily identified by altered phenotypes determinedby growth on agar plates or by simple assays and individualmutants and population samples can be indefinitely stored atminus80∘C allowing comparisons to be made across time pointsand between ancestral and evolved strains In particular thisallows testing of fitness changes where adaptive genotypesare defined as having a competitive fitness (W) advantageover the ancestorW can be calculated as the ratio of Malthu-sian parameters for continuously growing populations whenW gt 1 the mutant has a fitness advantage over the ancestorand is considered an adaptive genotype whenW = 1 the twostrains are neutral with respect to one another and whenWlt 1 the mutant is at a selective disadvantage [15ndash17]

If the possibility of HGT is excluded in experimentalpopulations the origin of all adaptive mutations must bethrough alterations to the ancestral bacterial genome (chro-mosome and accompanying plasmids) andmight range fromsmall-scale sequence changes affecting a single gene to largerrearrangements including deletions or duplications of wholeoperons or considerable portions of the genome These canbe identified through sequencing target genes or throughwhole genome resequencing and the underlying molecularbiology of individual genotypes can be further investigated interms of gene expression patterns regulatory networks andmetabolism in order to understand how adaptive mutationscan be mechanistically linked to altered phenotypes andfitness changes [18]

Although experimental microcosms tend to be physi-cally and chemically simple they can be manipulated tochange ecological opportunities competition environmentalconditions and selective pressures For example glass vialscontaining liquid growth medium can be incubated withconstant shaking to provide a homogeneous environmentor statically where spatial structure becomes important withthemicrocosm becoming heterogeneous and containing newniches at the air-liquid interface the liquid column and vialbottom [19] Nutrient type abundance and complexity aswell as the chemical environment (eg osmolarity O

2 and

pH) can all be manipulated through changes to the growthmediumThe small size low cost and reproducibility of suchmicrocosms mean that they can be used in large numberswith appropriate levels of replication allowing multifactorialexperimental design and including the ability to repeatexperiments using exactly the same initial conditions whenrequired

12 Some Molecular Aspects Underlying Bacterial Evolu-tion In comparison with highly specialized bacteria suchas pathogens and symbionts generalists have a high pro-portion of sensory and regulatory systems which allowthem to respond to a wide range of environmental fac-tors and opportunities These presumably arose throughdistant gene duplication and acquisition events and ofteninvolve multifunctional proteins linking sensory and signaltransduction domains which retain little homology beyondthe functional domains themselves While some regulatory

elements act to modify cellular response or homeostasisvia transcription others modify activity through secondarysignal molecules such as cyclic-di-GMP (bis-(31015840-51015840)-cyclicdimeric guanosine monophosphate) Cyclic-di-GMP is anintracellular signalling molecule that plays a central role inthe regulation of motility virulence and biofilm formationin many bacteria (for reviews see [20ndash24]) It is synthe-sised from GTP (guanosine-51015840-triphosphate) by DGCs (di-guanylate cyclases) and hydrolysed by specific PDEs (phos-phodiesterases) to guanosine monophosphate (GMP) or 51015840-pGpG (linearized di-GMP)which is subsequently hydrolysedto GMP by other hydrolases DGCs are characterised by theamino acid motif Gly-Gly-Asp-Glu-Phe referred to as theGGDEFdomain whilst PDEs include theGlu-Ala-Leu (EAL)or His-Asp-x-Gly-Tyr-Pro (HD-GYP) domains

Bacterial genomes tend to have multiple DGCs andPDEs which suggests that cyclic-di-GMP levels are regulatedthrough a complex signaling network integrating numerousenvironmental signals that control riboswitches transcrip-tion factors and enzyme activities including cellulose expres-sion The disruption of homeostasis through mutation of ahigh-level sensory-regulator systems can have a significantimpact on bacterial phenotype allowing significant fitnessleaps to occur rather than the expected smaller incrementalsteps Furthermore the duplication and divergence of reg-ulators can lead to significant reprogramming of regulatorynetworks [25] with transferred genes (xenologues) generallypersisting in genomes longer than duplicated genes (par-alogues) which tend to have more protein-protein interac-tions and regulators [8]

2 Adaptive Radiation of P fluorescens inStatic Microcosms

The soil and plant-associated fluorescent pseudomonadPseudomonas fluorescens SBW25 has been used as a modelbacterium in experimental evolution studies using simplemicrocosms [19] SBW25 was originally isolated from the leafof a sugar beet (Beta vulgaris) plant [26] and is capable ofcolonising a wide variety of crop plants and weeds Like otherfluorescent pseudomonads it is regarded as a benign plantgrowth-promoting rhizobacterium (ie one that grows onor around the roots of plants in the rhizosphere) HoweverSBW25 carries a defective pathogen-like type III secretionsystem [27] and expresses the virulence-associated cycliclipopeptide class surfactant viscosin [28]Many P fluorescensstrains including SBW25 produce soft rot-like symptomswhen colonising plant tissues following physical damagesuggesting that these soil and plant-associated bacteria arehighly competent colonists with opportunistic pathogenictendencies The ability to colonise new environments is acharacteristic associated with the pseudomonads and maybe enabled by relatively large genomes that include manysensory and regulatory elements [29ndash31] SBW25 sequenceswere initiallymaintained in a partial genomic database beforethe whole genome sequence was determined [30 32] andcomprehensive molecular analyses of regulatory pathwaysmetabolism and fitness are possible using techniques estab-lished for other pseudomonads In addition to experimental









2diffusing


















2







Timeline

Epoch

Genotypes

and niches

Events and

consequenceslowast







From the first day

Diversification








the liquid column




(Similarly)

(Similarly)











GMP

WspA

WspC WspF

WspDWspB

WspE

WspR

Innermembrane

+CH3 minusCH3

Cyclic-di-GMP


3























Acknowledgments


References








































































































2diffusing


















2







Timeline

Epoch

Genotypes

and niches

Events and

consequenceslowast







From the first day

Diversification








the liquid column




(Similarly)

(Similarly)











GMP

WspA

WspC WspF

WspDWspB

WspE

WspR

Innermembrane

+CH3 minusCH3

Cyclic-di-GMP


3























Acknowledgments


References







































































































2







Timeline

Epoch

Genotypes

and niches

Events and

consequenceslowast







From the first day

Diversification








the liquid column




(Similarly)

(Similarly)











GMP

WspA

WspC WspF

WspDWspB

WspE

WspR

Innermembrane

+CH3 minusCH3

Cyclic-di-GMP


3























Acknowledgments


References

































































































Timeline

Epoch

Genotypes

and niches

Events and

consequenceslowast







From the first day

Diversification








the liquid column




(Similarly)

(Similarly)











GMP

WspA

WspC WspF

WspDWspB

WspE

WspR

Innermembrane

+CH3 minusCH3

Cyclic-di-GMP


3























Acknowledgments


References

































































































GMP

WspA

WspC WspF

WspDWspB

WspE

WspR

Innermembrane

+CH3 minusCH3

Cyclic-di-GMP


3























Acknowledgments


References
















































































































Acknowledgments


References




































































































Acknowledgments


References





































































































































































































Documents

ReviewArticle - Abertay University · ReviewArticle A Mechanistic Explanation Linking Adaptive Mutation, Niche Change, and Fitness Advantage for the Wrinkly Spreader AndrewJ.Spiers