Biology of Moderately Halophilic Aerobic Bacteria

41 Pages·1998·0.37 MB·English

MICROBIOLOGYANDMOLECULARBIOLOGYREVIEWS,June1998,p.504–544 Vol.62,No.2
1092-2172/98/$04.0010
Copyright©1998,AmericanSocietyforMicrobiology
Biology of Moderately Halophilic Aerobic Bacteria
ANTONIOVENTOSA,1*JOAQU´INJ.NIETO,1ANDAHARONOREN2
DepartmentofMicrobiologyandParasitology,FacultyofPharmacy,UniversityofSeville,41012Seville,Spain,1and
DivisionofMicrobialandMolecularEcology,InstituteofLifeSciences,andtheMosheShiloMinervaCenter
forMarineBiogeochemistry,TheHebrewUniversityofJerusalem,91904Jerusalem,Israel2
INTRODUCTION.......................................................................................................................................................505
TAXONOMYANDPHYLOGENY...........................................................................................................................506
ECOLOGY...................................................................................................................................................................509
NumericalTaxonomyApproachestotheAnalysisofNaturalCommunitiesofModerately
HalophilicBacteria.........................................................................................................................................509
(i)Saltlakesandbrines....................................................................................................................................509
(ii)Salinesoils....................................................................................................................................................511
(iii)Coldsalinehabitats....................................................................................................................................512
(iv)Alkalinesalinehabitats..............................................................................................................................513
(v)Saltedfish,meat,andotherfoods.............................................................................................................513
(vi)Unusualhabitats.........................................................................................................................................513
MorphologicalandPhysiologicalDiversity.........................................................................................................513
MeasurementofInSituActivities........................................................................................................................514
MolecularApproachestotheElucidationoftheCommunityStructureofHalophilicBacteria
inHypersalineEnvironments............................................................................................................................514
CompetitionbetweenHalophilicBacteriaandArchaea....................................................................................514
BiogeochemicalImportance...................................................................................................................................515
Bacteriophages........................................................................................................................................................515
PHYSIOLOGY............................................................................................................................................................516
RequirementforSalt..............................................................................................................................................516
MinimalandDefinedMedia.................................................................................................................................516
TolerancetoHeavyMetalIons.............................................................................................................................517
InternalIonConcentrations..................................................................................................................................517
(i)Sodium............................................................................................................................................................519
(ii)Potassium......................................................................................................................................................519
(iii)Magnesium...................................................................................................................................................519
(iv)Calcium.........................................................................................................................................................519
(v)Manganese.....................................................................................................................................................519
(vi)Chloride........................................................................................................................................................519
IonPumpsintheCellMembranes......................................................................................................................519
TransportofOrganicCompounds.......................................................................................................................521
OrganicOsmoticSolutes.......................................................................................................................................521
(i)Aminoacids....................................................................................................................................................522
(ii)Glycinebetaine.............................................................................................................................................522
(iii)Ectoineandhydroxyectoine.......................................................................................................................523
(iv)Otherorganicosmoticsolutes...................................................................................................................523
(v)Distributionoforganicosmoticsoluteswithinmoderatelyhalophilicbacteria..................................523
(vi)Uptakeandactionofexogeneouslysuppliedglycinebetaine................................................................523
(vii)Accumulationofotherosmoticsolutesbytransportfromthemedium.............................................524
ENZYMES...................................................................................................................................................................524
CytoplasmicEnzymes.............................................................................................................................................525
StressProteins........................................................................................................................................................526
Membrane-BoundEnzymes...................................................................................................................................526
ExtracellularEnzymes............................................................................................................................................526
AminoAcidCompositionofProteinsfromModeratelyHalophilicBacteria.................................................527
CELLENVELOPES...................................................................................................................................................527
PolarLipids.............................................................................................................................................................527
(i)Salinivibriocosticola.......................................................................................................................................528
(ii)Halomonaselongata.......................................................................................................................................528
(iii)Halomonasisraelensis..................................................................................................................................528
*Correspondingauthor.Mailingaddress:DepartmentofMicrobi-
ology and Parasitology, Faculty of Pharmacy, University of Seville,
41012 Seville, Spain. Phone: [phone]. Fax: [phone]. E-
mail:[email protected].
504
VOL.62,1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 505
(iv)“Pseudomonashalosaccharolytica”..............................................................................................................528
(v)Othergram-negativehalophilicbacteria...................................................................................................529
(vi)Gram-positivemoderatehalophiles..........................................................................................................529
(vii)Adaptationofthepolarlipidcompositiontochangingsalinities.......................................................529
EffectofSaltConcentrationonFattyAcidComposition..................................................................................529
(i)Gram-negativehalophilicbacteria..............................................................................................................529
(ii)Gram-positivehalophilicbacteria.............................................................................................................530
GENETICS..................................................................................................................................................................530
GenomeAnalysisandPhysicalMaps..................................................................................................................531
NativePlasmidsandDerivedCloningVectors...................................................................................................531
GeneticTransfer.....................................................................................................................................................532
ReporterGenes........................................................................................................................................................533
IsolationofMutants...............................................................................................................................................534
GeneticBasisofOsmoregulation.........................................................................................................................534
BIOTECHNOLOGICALAPPLICATIONS.............................................................................................................535
FermentedFoods.....................................................................................................................................................535
Enzymes....................................................................................................................................................................535
CompatibleSolutes.................................................................................................................................................535
Polymers...................................................................................................................................................................536
DegradationofToxicCompounds........................................................................................................................536
OtherPotentialApplications.................................................................................................................................536
CONCLUSIONANDFUTUREDIRECTIONS......................................................................................................537
ACKNOWLEDGMENTS...........................................................................................................................................537
REFERENCES............................................................................................................................................................537
INTRODUCTION Theoccurrenceofnonpigmentedhalotolerantbacteriawas
probably first mentioned in 1919 by LeFevre and Round in
Comparedtotheextensiveliteratureonthephysiology,bio- their study of the microbiology of cucumber fermentation
chemistry, and ecology of the aerobic red halophilic archaea brines.Oneofthebacterialgroupsisolatedgrewin0to15%
(familyHalobacteriaceae),theaerobichalophilicbacteriahave NaCl, whereas other bacteria studied exhibited growth over
been relatively little studied. Research on the halophilic and therangeof5to25%(178).Anearlyclassicstudyofhalophilic
halotolerantbacteriaoftenseemstobelessglamorousthanthe bacteria is the work of Hof (127), who inoculated salt mud
studyofthearchaea,withtheiruniqueadaptations,including from a solar salt facility on Java onto a variety of media of
a highly saline cytoplasm, specialized salt-requiring proteins, differentsalinities.Inadditiontoredarchaealtypes,different
andtheuniquelight-drivenprotonandchloridepumpsbacte- types of white colonies were isolated, including endospore-
riorhodopsin and halorhodopsin (171). During early research containing Bacillus species able to grow at 24% NaCl. Using
on the microbiology of hypersaline environments, the halo- media containing between 12 and 18% salt, she isolated a
philicbacteriawereoftenneglected,eventhoughtheyinhabit Pseudomonas-type bacterium from salted beans preserved in
a wide range of habitats such as saline lakes, saltern ponds, brines varying in salt concentration from 6 to 29%. This or-
desert and hypersaline soils, and salted foods, a range much ganism, designated Pseudomonas beijerinckii, grew from 3 to
lessrestrictedthanthehabitatsinwhichthehalophilicarchaea 18%saltbutnotat0.5%,showingitsobligatehalophilicchar-
thrive(276,284,285). acter. To quote from Hof’s paper: “it may be concluded that
However, Kushner (168) clearly states: “Though they are most of the important groups of bacteria are able to live in
less exciting at first glance than the extreme halophiles the concentrationsuptoabout15%saltandthatmanygroupsare
moderately halophilic bacteria, and solute-tolerant microor- physiologicallyactiveevenatmuchhighersaltconcentrations.”
ganisms in general, pose quite sufficiently interesting ques- Todescribemicroorganismsaccordingtotheirbehaviorto-
tions,especiallythoseimpliedbytheirabilitytogrowoverwide ward salt, different classification schemes have been devised.
ranges of solute concentrations. Further work on these rela- Although several classifications or categories have been pro-
tively little-studied microorganisms may be expected to bring posed(274,329,351),themostwidelyusedisthatofKushner,
dividendsintheformofinsightontherelationofinternaland who defined moderate halophiles as organisms growing opti-
externalsoluteconcentrations,andonthestateofcell-associ- mallybetween0.5and2.5Msalt(168).Bacteriaabletogrow
atedionswithinthecytoplasm.Ifthelastdecadehasbeenthat in the absence of salt as well as in the presence of relatively
oftheextremehalophiles,wecanhopethatthenextonewill highsaltconcentrations(e.g.,8%inthecaseofStaphylococcus
seetheirmoremodest,moderatecousins(inthespiritualsense aureus)aredesignatedhalotolerant(orextremelyhalotolerant
only) take their proper place in the scientific canon.” The ifgrowthextendsabove2.5M).Ararecaseofabacteriumthat
moderately halophilic bacteria pose specific questions to the requires2Msaltatleast(optimalgrowthat3.4M),suchasis
scientist,manyofthemrelatedtotheiradaptabilitytoawide exemplified by the actinomycete Actinopolyspora halophila
range of salinities. Thus, species such as Salinivibrio costicola (95),isconsideredaborderlineextremehalophile(137,168).
andHalomonashalodenitrificansareabletogrowoverarange It should be pointed out that the salt requirement and tol-
of water activities between 0.98 (close to freshwater) to 0.86 erance of many species vary according to growth conditions
(closetosaturatedNaCl)(168).Thisinitselfisafeatthatmay such as temperature and medium composition. The growth
bemuchmoredifficulttoachievethantherigid,salt-requiring temperatureshouldbespecified,especiallyforthedefinitionof
metabolismofthehalophilicarchaea,whichlysethemoment thelowersaltrangeenablinggrowth.Thus,Marinococcushalo-
thesaltconcentrationintheirenvironmentdropsbelow10to philusgrowsatNaClconcentrationsaslowas0.01Mat20°C
15%. but at least 0.5 M is required at 25°C (228). Similarly, S. cos-
506 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV.
TABLE 1. Maincharacteristicsofmoderatelyhalophilicgram-negativemembersoftheHalomonadaceaea
H.elongata H.subglaciescola H.halodurans H.halmophila H.eurihalina H.halophila
Feature
(357) (74) (115) (54) (200,266) (269)
Morphology Rods Rods Rods Rods Shortrods Rods
Size(mm) ND 0.5–1.135–10 0.4–0.631.5–2.0 0.3–0.630.9–1.3 0.8–1.032.0–2.5 0.5–0.731.5–2.0
Pigmentation None Cream None Cream Cream Cream
Motility 1 1 1 1 2 1
Facultativeanaerobe 1 2 2 2 2 2
Oxidase 1 1 1 1 2 1
NaClrange(%) 3.5–20 0.5–20 3.5–20 0.5–20 5–20 2–30
NaCloptimum(%) 3.5–8 ND 8 ND 7.5 7.5
Temprange(°C) 15–45 0–25 4–37 ND 15–45 15–45
pHrange 5–9 5–9 5.5–8.5 ND 5–10 5–10
Acidproductionfrom:
Arabinose 2 2 1 ND 2 2
Glucose 1 1 1 1 2 1
Lactose 2 1 2 2 2 2
Trehalose ND ND ND ND 2 ND
Mannitol 2 2 2 1 2 2
Hydrolysisof:
Gelatin d 2 2 2 1 2
Casein 2 d ND 2 2 2
Starch 2 2 2 2 2 2
Esculin 2 2 1 2 1 1
Tween80 2 2 ND 2 1 2
DNA 2 2 ND 1 1 2
HSproduction 2 2 2 ND 1 1
Ni2tratereduction 1 1 2 2 1 1
Nitritereduction 1 2 2 2 2 2
Phosphatase ND 2 2 2 1 1
Typestrain ATCC33173 UQM2927 ATCC29686 ATCC19717 ATCC49339 CCM3662
Habitat Salterns Antarcticsalinelakes Estuarinewater DeadSea Salinesoils,salterns Salinesoils
G1Ccontent(mol%) 60.5 60.9–62.9 63.2 63 59.1–65.7 66.7
Phylogeneticbranch GammaProteo- GammaProteo- GammaProteo- GammaProteo- GammaProteo- GammaProteo-
bacteria bacteria bacteria bacteria bacteria bacteria
a1,positive;2,negative;d,differsamongstrains;ND,notdetermined.
ticolacangrowbetween0.5and4MNaClat30°Cbutcangrow Volcaniella, Flavobacterium, Paracoccus, Pseudomonas, Halo-
downto0.2Mat20°C(168). vibrio,orChromobacterium),phenotypicandphylogeneticdata
supporttheircloserelationship,andtheyarecurrentlyinclud-
TAXONOMYANDPHYLOGENY ed in the family Halomonadaceae as members of two genera:
HalomonasandChromohalobacter(53,76).Table1showsthe
Moderately halophilic bacteria constitute a heterogeneous featuresthatdifferentiatethevalidlypublishedmoderatelyhalo-
physiologicalgroupofmicroorganismswhichbelongtodiffer-
philicspeciesincludedinthesetwogenera.
entgenera.Ourcurrentknowledgeofthetaxonomicstatusof
Besides members of the family Halomonadaceae, several
these bacteria contrasts with early studies, since in 1980 only
othergram-negativestrictlyaerobicorfacultativelyanaerobic
six moderately halophilic species were included in the Ap-
specieshavebeendescribedasmoderatehalophilesbelonging
provedListsofBacterialNames(312)andmoststrainsusedin
to genera that include nonhalophilic species as well, such as
physiologicalandbiochemicalstudieswereisolatedfromsalt-
Pseudomonas,Flavobacterium,orSpirochaeta,whileothersare
ed cured foods or unrefined salt or were even laboratory cul-
placedingenerarepresented,atleastuntilnow,exclusivelyby
turecontaminants.ThesemoderatehalophileswereVibrio(Sal-
halophilicspecies:Salinivibrio,Arhodomonas,orDichotomicro-
inivibrio)costicola(314),Micrococcus(Nesterenkonia)halobius
bium. The differential features of these species are shown in
(237), Paracoccus (Halomonas) halodenitrificans (159), Flavo-
Table2.
bacterium(Halomonas)halmephilum(60),Planococcus(Mari-
Threeorganismshavebeenextensivelyusedinphysiological
nococcus)halophilus(228),andSpirochaetahalophila(103).
andbiochemicalstudiesdealingwiththemechanismsofhalo-
Duringthelastdecade,theextensivestudiesonhypersaline
environmentsthathavebeencarriedoutinmanygeographical adaptation and adaptability: Salinivibrio costicola, Halomonas
areas have permitted the isolation and taxonomic character- elongata,andHalomonasisraelensis.
ization of a large number of moderately halophilic species. S.costicolawasoriginallyisolatedfromribbonesinasample
Thus, moderate halophiles are represented by several meth- of Australian bacon (91, 314). Similar strains have been iso-
anogenicarchaeaaswellasstrictlyanaerobicbacteriathathave latedfromsalternpondsnearAlicante(96)andotherlocations
beenreviewedrecently(182,232)andarenotincludedinthis inSpainandtheCanaryIslands(91).
article.However,mostspeciesaregram-negativeorgram-posi- H.elongatawasisolatedfromasolarsaltfacilityonBonaire,
tive aerobic or facultatively anaerobic moderately halophilic Netherlands Antilles. It is an extremely versatile organism,
bacteria(340,341).Althoughsomegram-negativespecieswere abletogrowataverywiderangeofsaltconcentrations.Itcan
considered members of different genera (Halomonas, Deleya, also grow anaerobically with nitrate as the electron acceptor,
VOL.62,1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 507
TABLE 1—Continued
H.salina H.halodenitrificans H.variablis H.canadensis H.israelensis H.pantelleriense Chromohalobacter
(53,336) (53,159) (53,65) (128) (128) (290) marismortui(344)
Shortrods Shortrods Curvedrods Rods Rods Rods Rods
0.7–0.832.0–2.5 0.5–0.930.9–1.2 0.5–0.831.0–3.0 ND ND 0.4–0.731.4–2.6 0.6–1.031.5–4.0
Yellowishorcream Cream Cream White Cream Cream-pink Brown-yellow
2 2 2 1 ND 1 1
2 2 2 2 2 2 2
1 1 1 2 2 2 2
2.5–20 3–20 7–28 3–25 3.5–20 1.2–15 1–30
5 5–9 10 8 8 10 10
15–40 5–32 15–37 15–30 15–45 10–44 5–45
6–10 ND 6.5–8.4 5–9 5–9 7.5–11 5–10
2 ND ND ND ND ND 1
2 2 ND ND ND ND 1
2 ND ND 2 2 ND 1
2 ND ND ND ND ND 1
2 ND ND ND ND ND ND
2 2 2 2 ND 2 2
2 ND ND 2 ND 2 2
2 2 2 2 ND 2 2
2 2 1 ND ND ND 2
2 2 ND ND ND ND 2
2 ND ND ND ND ND 2
1 2 2 2 2 ND 2
1 1 2 1 1 1 1
2 1 2 2 2 ND 2
2 2 ND ND ND ND 2
ATCC49509 ATCC13511 DSM3051 ATCC43984 ATCC43985 DSM9661 ATCC17056
Salinesoils,salterns Meat-curingbrines GreatSaltLake Unknown(con- DeadSea Alkalinesalinelake DeadSea,salterns
taminant)
60.7–64.2 64–66 61 57 64 65 62.1–64.9
GammaProteo- GammaProteo- GammaProteo- ND ND GammaProteo- GammaProteo-
bacteria bacteria bacteria bacteria bacteria
formingnitrite,andithasalsobeenreportedtogrowfermen- permittedadeterminationofthephylogeneticpositionofmost
tatively on glucose (357). However, glucose fermentation was moderately halophilic bacteria. During last decade, the old
notconfirmedinlaterstudies(83). technique of comparison of 16S rRNA oligonucleotide cata-
H.israelensis(previouslydesignatedstrainBa )wasoriginal- logs showed that Spirochaeta halophila belongs to the spiro-
1
lyobtainedfromunrefinedsolarsaltobtainedfromtheDead chetephylumandthatsomeHalomonasandDeleyaspeciesare
Sea(272,273)andwasonlyrecentlyassignedaspeciesname members of the gamma subclass of the Proteobacteria (341).
(128). Theywereplacedinanewfamily,Halomonadaceae(76).More
The gram-positive moderately halophilic aerobic bacteria, recently,complete16SrRNAsequenceanalysisconfirmedthat
withtheexceptionoftwoBacillusspecies,belongtogenerathat Spirochaeta halophila is within the Spirochaeta cluster of the
include only species with halophilic requirements: the genera spirochetephylum,relatedtoSpirochaetaisovalerica,S.litora-
Halobacillus, Marinococcus, Salinicoccus, Nesterenkonia, and lis, S. bajacaliforniensis, and S. aurantia (average similarity,
Tetragenococcus. The validly described species currently ac- 87.4%) (253). In addition, several studies have identified the
cepted as moderate halophiles and their characteristics are phylogeneticpositionofmostgram-negativemoderatelyhalo-
showninTable3.Finally,therearesomemoderatelyhalophil- philic aerobic species currently described. Members of the
ic actinomycetes that have been recently isolated from differ- generaHalomonas,Deleya,Halovibrio,andVolcaniella,aswell
entsalinesoilsamplesandhavebeencharacterizedasspecies as Paracoccus halodenitrificans, form a monophyletic group
of the genera Actinopolyspora or Nocardiopsis. Table 4 shows within the gamma subclass of the Proteobacteria (53, 56, 200,
the differential characteristics of these four moderately halo- 204,290).Thelevelsof16SrRNAsequencesimilarityamong
philicspecies. thesespeciesrangedfrom91.5to100%;althoughseveralsub-
Besides these moderately halophilic species that have been groups,whichmightrepresentseparategenera,wereresolved,
characterizedtaxonomicallyand,accordingtotherulesofthe theycouldnotbedifferentiatedonthebasisofphenotypicor
code of nomenclature of bacteria, are considered validly de- chemotaxonomicfeatures.Forthesereasons,DobsonandFranz-
scribed species, there are several other moderately halophilic mann (53) proposed placing all members of the above four
aerobic bacteria that have been used for other purposes, in- genera and P. halodenitrificans in a single genus, the genus
cludingphysiological,biochemical,andbiotechnologicalstud- Halomonas, and emended the description of the family Hal-
ies,buthavenotbeenstudiedtaxonomicallyindetail.Typical omonadaceae.ThisfamilynowcomprisesthespeciesofHalo-
examplesare“Pseudomonashalosaccharolytica”(120,121)and monasandZymobacterandthemoderatehalophile,originally
“Micrococcusvarianssubsp.halophilus”(241). isolated from the Dead Sea, Chromohalobacter marismortui
Recentstudiesbasedon16SrRNAsequenceanalysishave (200).Allhave15signaturecharacteristicsintheir16SrRNA
5
0
8
V
E
N
T
O
TABLE 2. Maincharacteristicsofotheraerobicorfacultativelyanaerobicgram-negativemoderatelyhalophilicbacteriaa SA
E
Salinivibriocosticola Pseudomonas Flavobacterium Flavobacterium Arhodomonas Spirochaeta Dichotomicrobium T
Feature (91,201) halophila gondwanense salegens aquaeolei halophila thermohalophilum A
(65) (52) (52) (4) (103) (124) L
.
Morphology Curvedrods Rods Rods Rods Shortrods Helicoidal Prosthecate
Size(mm) 0.531.5–3.2 0.8–1.031.5–5.0 0.4–0.831.7–11.7 0.5–0.831.2–11.5 0.8–1.032.0–2.5 0.4315–30 0.8–1.830.8–2.0
Pigmentation Cream Reddish-brown Orange Yellow None Red Reddish-brown
Motility 1 1 2 2 1 1 2
Facultativeanaerobe 1 2 2 2 2 1 2
Oxidase 1 1 1 1 1 ND 1
NaClrange(%) 0.5–12 0.1–20 0–5 0–20 6–20 0.5–8 0.8–22
NaCloptimum(%) 10 5 5 5 15 5 8–14
Temprange(°C) 5–45 4–37 0–30 ND 20–45 25–40 20–60
pHrange 5–10 4.5–9.6 5–9 5–9 6–8 5.8–9.5
Acidproductionfrom:
Arabinose 2 ND 2 1 2 ND ND
Glucose 1 ND 2 1 2 1 2
Lactose 2 ND 2 1 2 ND ND
Trehalose 1 ND ND ND 2 ND ND
Mannitol 1 ND ND 1 2 ND ND
Hydrolysisof:
Gelatin 1 1 d 1 2 ND 2
Casein 1 1 2 2 2 ND 2
Starch 2 2 1 1 2 ND 2
Esculin 1 1 1 1 2 ND ND
Tween80 d ND 1 1 1 ND ND
DNA d ND 1 1 2 ND 2
H Sproduction d 2 2 2 2 ND 2
Ni2tratereduction 2 2 2 1 1 1 2
Nitritereduction 2 2 2 2 2 ND 2
Phosphatase 2 ND 1 1 2 ND ND
Typestrain NCIMB701 DSM3050 DSM5423 DSM5424 ATCC49307 ATCC29478 DSM5002
Habitat Salterns GreatSaltLake HypersalineAntarctic HypersalineAntarctic Petroleumreservoir SolarLake SolarLake
lake lake producingfluid
G1Ccontent(mol%) 49.4–50.5 57.0 35–39 39–41 67 62 62–64 M
Phylogeneticbranch GammaProteobacteria ND Flavobacterium- Flavobacterium- GammaProteobacteria Spirochetes ND IC
R
Bacteroides Bacteroides O
B
a1,positive;2,negative;d,differsamongstrains;ND,notdetermined. IOL
.
M
O
L
.
B
IO
L
.
R
E
V
.
VOL.62,1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 509
sequences, including a distinctive cytosine residue at position the moderate halophiles, these authors concluded that the
486(53).Arhodomonasaquaeoleirepresentsadeeplybranch- alkaliphile phenotype is also polyphyletic and might have
ing lineage in the gamma subclass of the Proteobacteria, most evolvedmanytimes(58).
closelyrelatedtopurplesulfurbacteria(particularlyspecies
of the genera Ectothiorhodospira and Chromatium). The 16S
ECOLOGY
rRNAsequenceanalysissupportstheplacementofthissingle
speciesinaseparategenus(4).
NumericalTaxonomyApproachestotheAnalysisofNatural
Veryrecently,thephylogeneticpositionofsix(Salini)vibrio
CommunitiesofModeratelyHalophilicBacteria
costicola strains revealed that this moderate halophile consti-
tutesamonophyleticbranchthatisdistinctfromotherVibrio (i) Salt lakes and brines. The communities of moderately
species and from other species belonging to the gamma sub- halophilicbacteriainthalassohaline(seawater-derived)hyper-
class of the Proteobacteria (202). Since other phenotypic and saline environments, such as saltern ponds for concentrating
genotypic data supported these differences, placement of this seawater, may to a large extent resemble the communities
species in a separate genus, Salinivibrio, has been proposed present in seawater. This is not too surprising, since many
(201). marine bacteria have a broad salt tolerance. It was even re-
The 16S rRNA sequences of Flavobacterium gondwanense ported that the majority of 30 isolates of marine aerobic het-
andF.salegens,twomoderatehalophilesisolatedfromahyper- erotrophicbacteriatestedcouldgrowatupto20%NaCland
saline Antarctic lake, contain the definitive flavobacterial sig- thatsomecouldevenmultiplyinmediacontaining30%NaCl
natures that unequivocally place them in the Flavobacterium- (72). Moderately halophilic bacteria could also be enriched
Bacteroidesphylum(52).Thesespeciesclusterwithagroupof fromseawaterbygradualsalinityincreases:whenseawaterwas
organismsthatcontainsthetypespeciesofthegenusFlavobac- periodicallyamendedwithsaltandnutrients,moderatelyhalo-
terium, F. aquatile (with 89 and 90% sequence similarity be- philicbacteriaoutcompetedtheslightlyhalophilicmarinebac-
tweenthemandthisspecies)(52). teria, which completely disappeared above 15% salt (347). A
Recent studies have determined the phylogenetic relation- varietyofhalophilicbacteriawerealsoisolatedfromseasands
shipsofmoderatehalophileswithinthegram-positivebranch. and seaweeds (235). Thus, the sea contains many moderately
Farrow et al. (64) showed that Marinococcus halophilus (for- halophilicoratleastextremelyhalotolerantbacteria.Inastudy
merlyPlanococcushalophilus)formsadistinctlineofdescent of Spanish saltern ponds of intermediate salinity (between 15
andisonlydistantlyrelatedtothegeneraPlanococcus,Sporo- and 30% sea salts) (Alicante on the Mediterranean coast,
sarcina, and Bacillus. The 16S rRNA sequence data confirm HuelvaontheAtlanticcoast),thedominanttypesofcolonies
theplacementofM.halophilusinaseparategenus.Inaddition, developing on agar plates were assigned by numerical taxon-
theyindicatedthatSporosarcinahalophila,anendospore-form- omytothegeneraSalinivibrio,thePseudomonas-Alteromonas-
ing motile gram-positive moderate halophile, was not closely Alcaligenes group, Acinetobacter, and Flavobacterium (284–
related to Sporosarcina ureae, the type species of this genus. 286).Mostoftheseisolatesshouldprobablybereclassifiedin
LaterstudiespermittedtheplacementofS.halophilainanew the family Halomonadaceae. Most isolates grew optimally at
genus, Halobacillus, as H. halophilus, closely related to oth- about10%salts(theconcentrationthatalsoyieldedthelargest
er moderately halophilic species isolated from the Great Salt number of CFU on agar plates) and could be found at salt
Lake, H. litoralis and H. trueperi (315). This study also con- concentrations up to about 25%. (Salinivibrio species domi-
firmed the placement of Salinicoccus roseus in a separate nated below 15% salt, while bacteria assigned to the Pseudo-
genus, since it constitutes a deep branch not closely related monas-Alteromonas-Alcaligenes group were especially abun-
toothergram-positivebacteria.Bacillussalexigensisclosely dantabove15%.FlavobacteriumandAcinetobacterwerefound
related to Bacillus pantothenticus, a species that belongs to in smaller numbers and were evenly distributed up to 30%,
phylogeneticgroupIofthegenusBacillus,aswellastoHalo- while gram-positive cocci were found mostly above 25% salt
bacillus halophilus, H. litoralis, and the halotolerant species (286).Mostofthe140isolatesfromtheHuelvasalternsclus-
Bacillusdipsosauri(89).Tetragenococcusmuriaticus,arecently teredineightphenons,twoofwhichwereidentifiedasHalo-
describedspeciesisolatedfromatraditionallyfermentedJap- monas(Deleya)(53),fourresembledS.costicola,andtheoth-
anese fish sauce, is closely related to the halotolerant species ersweretentativelyassignedtothegeneraFlavobacteriumand
Tetragenococcus halophilus (formerly Pediococcus halophilus), Acinetobacter (187). Similar Acinetobacter-like bacteria (prob-
whichshowedacloserphylogeneticrelationshiptootherlactic ablytobeassignedtotheHalomonadaceae)havebeenfound
acidbacteriaoftheenterococciandlactobacillithantopedio- inotherhypersalinehabitats(265).
cocci(304). Numerical taxonomic studies have been performed in in-
Whileallthesemoderatelyhalophilicspeciesbelongtothe land, athalassohaline salterns near Granada, Spain (39, 41,
low-G1C group of the gram-positive phylum, only Nesteren- 274),andChile(262).TheLaMal´asalternnearGranadaisfed
konia halobia (formerly Micrococcus halobius) is within the bybrinesfromasubterraneanwellwhichislowerinCl2and
high-G1C group (218, 316), constituting a cluster that is higher in Mg21, Ca21, and K1 than seawater. Of the 174
clearlyseparatefromotherspeciesofthegeneraMicrococcus, strainsisolated,74wereassignedtothegenus(Salini)vibrio,22
Arthrobacter,andKocuria. wereassignedtoAlteromonas,43wereassignedtoHalomonas
Therecentphylogeneticstudiesofmoderatehalophileshave (Deleya) (53), 7 were assigned to Acinetobacter, 13 were as-
been based on the comparison of 16S rRNA sequence data. signedtoPseudomonas,and9wereassignedtoFlavobacterium
Thesestudieshavebeenveryhelpfulforimprovingtheclassi- (37). An even more unusual inland saltern is the Salar de
ficationofmoderatehalophilesaccordingtoanatural(phylo- Atacama, Chile, located in the Atacama desert at 2,700 m
genetic) approach. In particular, results show that they are abovesealevel.Themostabundantisolatesobtainedfromthis
representedinmanyofthemajorbacterialphyla:spirochetes, environment were (Salini)vibrio strains, followed by Acineto-
Proteobacteria,Flavobacterium-Bacteroides,andlow-G1Cand bacter,Marinomonas,andAlteromonas(262).
high-G1Cgram-positiveorganisms.ArecentstudyofAfrican LakeAssalinDjibouti(FrenchSomaliland)contains27.7%
soda lakes showed a wide phylogenetic diversity within the salts in its surface layers, increasing to 39.8% at a depth of
alkaliphilic(andinsomecaseshalophilic)isolates(58).Aswith 20 m. Of the 164 isolates obtained, 11 were moderate halo-
510 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV.
TABLE 3. Maincharacteristicsofaerobicgram-positivemoderatelyhalophilicbacteriaa
Halobacillus Bacillus
Feature
H.halophilus H.litoralis H.trueperi B.halophilus B.salexigens
(33,315) (315) (315) (342) (89)
Morphology Coccoid Rods Rods Rods Rods
Size(mm) 1.0–2.032.0–3.0 0.7–1.132.0–4.5 0.7–1.432.0–4.5 0.5–1.032.5–9.0 0.3–0.631.5–3.5
Pigmentation Orange Orange Orange None None
Sporeshape S E/S E/S E E
Sporeposition C/ST C/ST C/ST C ST/C
Sporangiumswollen 2 ND ND 2 1
Motility 1 1 1 1
Facultativeanaerobe 2 2 2 2 2
Oxidase 1 1 1 1 1
NaClrange(%) 2–20 0.5–25 0.5–30 3–30 7–20
NaCloptimum(%) 10 10 10 15 10
Temprange(°C) 15–40 10–43 10–44 15–50 15–45
pHrange 7–9 6–9.5 6–9.5 6–8 6–11
Acidproductionfrom:
Arabinose 2 ND ND 2 ND
Glucose 2 1 1 1 1
Lactose ND ND ND 2 2
Trehalose 2 1 1 1 2
Mannitol 2 1 2 2 1
Hydrolysisof:
Gelatin 1 1 1 2 1
Casein 1 2 2 2 1
Starch 1 2 2 2 2
Esculin 2 2 2 1 1
Tween80 2 2 2 ND 2
DNA 1 1 1 1 1
H Sproduction ND ND ND 2 1
Ni2tratereduction 2 2 2 2 2
Nitritereduction 2 2 2 2 2
Phosphatase d 2 2 2 d
Voges-Proskauer 2 2 2 2 2
Menaquinonesystem MK-7 ND ND MK-7 ND
Cellwalltype Orn–D-Asp Orn–D-Asp Orn–D-Asp m-Dpm m-Dpm
Typestrain DSM2266 DSM10405 DSM10404 ATCC49085 ATCC700290
Habitat Salinesoils,salterns GreatSaltLake GreatSaltLake Unknown(rotting Salterns,salinesoils
woodfromseawater)
G1Ccontent(mol%) 40.1–40.9 42 43 51.5 36.3–39.5
Phylogeneticbranch Low-G1Cgram- Low-G1Cgram- ND ND Low-G1Cgram-
positive positive positive
aS, spherical; E, ellipsoidal; C, central; ST, subterminal. 1, positive; 2, negative; d, differs among strains; ND, not determined. MK-9, MK-8, MK-7, MK-6,
menaquinonewithnine,eight,seven,andsixisopreneunits,respectively.m-Dpm,meso-diaminopimelicacid.
philes requiring 3 to 15% salt, 7 were extremely halophilic plexmediumwith5,13,and20%NaClyielded3.63105,9.33
archaea,2didnottoleratesalt,andthegreatmajoritywere 105, and 2.4 3 106 CFU/ml, respectively, with surface water
slightly halophilic, requiring 1 to 5% NaCl (16). The authors fromthenortharmofthelake(22%salt),and7.53105,1.13
concludedthat“itisclearthatthebacteriawere,onthewhole, 105, and 1.5 3 103 cells/ml, respectively, when surface water
common bacteria belonging to the same species which are from the south arm (8.5% salt) served as the inoculum (65).
currentlyisolatedfromlargeandsmallriversandfromseawa- The data presented do not allow a differentiation between
ter.”Thisresultisunexpectedinviewofthehighsaltconcen- colorlessbacteriaandredarchaea,butitwasstatedthatmod-
trationsprevailinginLakeAssalandthehightemperatureof erately halophilic bacteria make up the predominant popula-
the water (33 to 34°C), which seem more suitable to the de- tion in the south arm whereas extremely halophilic archaea
velopmentofextremelyhalophilicarchaeathanofmarine-type predominateinthenortharm.TheGreatSaltLakeyieldeda
slightly or moderately halophilic bacteria. Regretably, addi- number of new species: Pseudomonas halophila, Halomonas
tionalstudiesofthisinterestingbutpoorlyaccessibleenviron- variabilis (53, 65), Halobacillus litoralis, and Halobacillus true-
menthavenotbeenreported. peri(315).
Asadlyneglectedhuntinggroundforinterestingnewtypes Althoughhalophilicbacteriacaneasilybeisolatedfromthe
ofmoderatelyhalophilicbacteriaistheGreatSaltLake,Utah, Dead Sea by means of enrichment cultures (242, 349), no
whichhasseendrasticchangesinitssalinityduringthelastde- systematicstudieshavebeenperformedontheirabundancein
cades. Most studies of its microbiology have centered around thisextremelyhypersaline(presentlyaround34%),athalasso-
the red halophilic archaea and the Dunaliella communities haline(around1.8MMg21,1.7MNa1,0.4MCa21,and0.14
(244,258,261).Attemptstoenumerateviablebacteriaincom- M K1) chloride lake. The first prokaryotes isolated from the
VOL.62,1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 511
TABLE 3—Continued
Marinococcus Salinicoccus
Nesterenkoniahalobia Tetragenococcus
M.halophilus M.albus S.roseus S.hispanicus (218,316) muriaticus(304)
(111,189) (111) (344) (188,345)
Cocci Cocci Cocci Cocci Cocci Cocci
1.0–1.2 1.0–1.2 1.0–2.5 1.0–2.0 ND 0.5–0.8
Yellow-orange None Pinkred Reddish-orange Noneoryellow White
2 2 2 2 2 2
2 2 2 2 2 2
2 2 2 2 2 2
1 1 2 2 2 2
2 2 2 2 2 1
2 1 1 1 1 2
2–25 2–25 0.9–25 0.5–25 3–25 1–25
10 10 10 10 10 7–10
15–37 15–37 15–40 15–37 20–40 15–40
ND ND 6–9 5–9 5–10 5–9.6
2 2 2 2 2 2
1 2 2 1 1 1
2 2 2 2 2 2
1 2 2 2 2 1
1 2 2 1 2 1
1 2 1 1 1 ND
1 2 1 2 1 ND
2 2 1 2 1 ND
1 2 2 1 1 ND
2 2 1 2 2 ND
2 1 1 1 1 ND
2 2 2 2 2 ND
2 1 2 1 2 2
2 2 2 1 2 2
2 2 1 d 2 ND
2 2 2 2 2 ND
MK-7 MK-7 MK-6 MK-6 MK-9,MK-8,MK-7 ND
m-Dpm m-Dpm L-Lys–Gly5 L-Lys–Gly5 L-Lys–Gly–L-Glu Lys–D-Asp
ATCC27964 CCM3517 ATCC49258 ATCC49259 ATCC21727 JCM10006
Salterns,salinesoils Salterns Salterns Salterns,salinesoils Salterns Fermentedsquidliver
sauce
46.4 44.9 51.2 45.6–49.3 ND 36.5
Low-G1Cgram- ND Low-G1Cgram- ND High-G1Cgram- ND
positive positive positive
lake(whichatthetimecontained28to29%salt)wereprob- peartoyieldmostlyhalotolerantratherthanhalophilicmicro-
ably colorless bacteria (362). Some of the early isolates were organisms, presumably reflecting adaptation to periodic epi-
characterizedfurtheranddescribedasChromohalobacter(orig- sodes of relatively high dilution (267, 268). One early study
inally Chromobacterium) marismortui (343), “Pseudomonas statedthatmicroorganismsmaybeunabletomultiplyinsaline
halestorgus” (subsequently lost), and Halomonas (originally soil and that the microbiota of saline soil habitats are passive
Flavobacterium)halmophila(54,60,350).Anothercolorless inhabitants brought by the wind. In this study, soils near the
isolatefromtheDeadSeaisHalomonasisraelensis(alsoknown Red Sea, with a salt content varying from 25 to 30% on the
asstrainBa )(128,170). surfaceto1.5to2%atadepthof50cm,wereexamined.The
1
Subterraneanbrineshavealsobeensubjecttomicrobiolog- highest bacterial counts were obtained in surface soil, using
ical studies. Several euryhaline microorganisms belonging to mediawithoutNaCl.Thenumbersobtainedweremuchsmall-
the Halomonadaceae, possibly including some novel species, erthanthosecommonlyfoundinnonsalinesoils.Theauthors
were isolated from Permian underground salt formations in stated that “the accepted assumption about the widespread
the United States which were penetrated by meteoric waters distribution of salt-resistant and halophilic microorganisms in
which slowly solubilize the salt (356). A subterranean brine salinesoilsrequiresreconsideration”(116).
associatedwithanoilfieldinOklahomayieldedArhodomonas Later studies have unequivocally confirmed the abundance
aquaeolei, an aerobic, organic acid-metabolizing, gram-nega- ofhalophilicbacteriainsalinesoils.Thespeciescompositionin
tive,motilerod,growingintherangeof6to20%NaClwithan soilsdiffersgreatlyfromthatoftheaquaticenvironmentsdis-
optimumof15%(4). cussedabove:whileSalinivibrioaboundsinsaltlakesandsalt-
(ii) Saline soils. The soil habitat is inherently inhomoge- erns,thedominanttypesencounteredinsalinesoilsbelongto
neous, and it can be expected that a wide range of salinities genera such as Bacillus, Pseudomonas, Alcaligenes (last two
might be present in any one saline soil (101). Saline soils ap- probably to be reclassified as members of the Halomonada-
512 VENTOSA ET AL. MICROBIOL.MOL.BIOL.REV.
TABLE 4. Maincharacteristicsofmoderatelyhalophilicactinomycetespeciesa
Actinopolyspora Nocardiopsis
Feature
A.mortivallis(374) A.iraquiensis(292) N.lucentensis(371) N.halophila(6)
Sporechainmorphology Short(nomorethan10) Short(nomorethan15) Short Verylong
Pigmentation Yellowishwhite Yellow-brownish Yellowishbrown Yellow-coralred
Spore Ovaltocylindrical Spheroidal Cylindrical Cylindrical
NaClrange(%) 5–30 5–20 ND 5–20
NaCloptium(%) 10–15 10–15 5–10 15
Temprange(°C) 10–50 16–40 ND 16–40
Acidproductionfrom:
Glucose 1 ND 1 2
Mannitol 2 1 1 1
Xylose 1 2 2 1
Nitratereduction 2 ND 1 ND
Hydrolysisof:
Gelatin 1 ND 1 ND
Casein 1 ND 1 ND
Esculin ND 2 1 2
Starch ND 1 1 2
Tyrosine ND 1 1 2
Tween80 1 1 ND 1
Urea 2 2 2 1
Xanthine 1 1 1 1
Menaquinonesystem MK-9(H),MK-10(H) MK-9(H) MK-10(H) MK-10(H,H)
4 4 4 8 6 8
Cellwalltype IV IV III III
Phospholipidtype PIII PIII PIII PIII
Mycolicacids 2 2 2 2
Typestrain JCM7550 A.S.4.1193 DSM44048 A.S.4.1195
Habitat Salinesoil Salinesoil Salinesoil Salinesoil
a1,positive;2,negative;ND,notdetermined.MK-10(H,H,andH),octa-,hexa-,andtetrahydrogenatedmenaquinoneswith10isopreneunits;MK-9(H),
8 6 4 4
tetrahydrogenatedmenaquinonewith9isopreneunits.
ceae), and Micrococcus (possibly Nesterenkonia) (268, 285). A soils in Death Valley (Calif.), Alicante, and Iraq (6, 291, 370,
salinesoilnearAlicante,Spain,withaCl2contentbetween2.4 373) suggests that a wealth of interesting unknown halophilic
and 12.7% yielded a high proportion of nonmotile rods on microorganismsmaybepresentinthesesoils.
mediacontaining10or20%salt;thistypeofbacteriumisonly (iii)Coldsalinehabitats.Extensivemicrobiologicalstudies
rarely isolated from aquatic hypersaline environments (265). intheAntarctic,especiallythecoldsalinelakesintheVestfold
The most abundant types were assigned to the genera Alcali- HillsregionandthesalinesoilsoftheDryValleys,havecon-
genes,Acinetobacter,Flavobacterium,andPseudomonas.Many tributedsomeinterestinginsightsintotheextremeconditions
halotolerant gram-positive bacteria were also found. Faculta- underwhichhalophilicbacteriamayoccurandthrive.
tive anaerobes were rare. Most isolates required moderately The Vestfold Hills region is a coastal, ice-free area in east
highsalt(5to10%)foroptimalgrowthbutwerealsoableto Antarctica, which contains in excess of 300 lakes and ponds.
growatlowsalinities(0.5to2%)(267).Thisprobablyreflects These are relics of seawater catchments isolated some 6,000
theecologicaldifferencebetweenwaterandsoilenvironments, years ago by uplift and trapped in valleys and depressions.
withwaterbeingrelativelyhomogeneousandconstantandsoil Someoftheselakesarehypersaline;themostsalinelakeshave
being heterogeneous and affected by factors such as rainfall a total salt concentration of up to 28%. Salinity may show
(267,268).Astudywasmadeoftherhizospheresoilnearxero- seasonalvariationsduetomeltstreaminfluxandicecovermelt
phytic plants growing in hypersaline soils (5 to 10.7% NaCl) in the Austral summer. The best studied is Organic Lake, a
near Alicante, showing that the range of salt concentrations meromictic lake with a maximum depth of 7.5 m. The lake is
allowing growth of the organisms isolated did not correlate stratified,withsaltconcentrationsincreasingfrom0.8to21%,
withthesalinityofthesoilfromwhichtheywereisolated.Most andisanoxicbelowadepthof4to5m.Theicecoverexcludes
isolates showed salt optima between 5 and 15% NaCl, but wind-induced turbulence throughout winter. Thermal profile
about half of the strains also grew at 0.9% NaCl. Plating on andtheincreasingsalinitywithdepthpreventturnoverinthe
agar media containing 10% salt yielded mostly gram-positive ice-free summer period. Temperatures range from 214 to
rods, whereas gram-negative rods dominated between 10 and 115°C(73,195,196).
20% salt and Gram-positive cocci developed above 20% salt. Many strains of moderate halophiles, belonging to genera
The gram-positive bacteria thus isolated were assigned to the includingHalomonas,Flavobacterium,andCytophaga,wereiso-
genera(inorderofabundance)Bacillus,Micrococcus,Arthro- latedfromthelake.Hexadecane-andphenanthrene-degrading
bacter, Staphylococcus, Planococcus, Corynebacterium, Nocar- bacteriawerealsofound(196).Mostisolateswereabletogrow
dia,andActinomyces(268). from0.5to20%NaClandattemperaturesaslowas0to5°C
Salinesoilshavebeensomewhatneglectedcomparedtohy- (55, 74). The species isolated include Halomonas subglacies-
persaline aquatic environments. The recent isolation of novel cola,growingatsaltconcentrationsbetween0.5and20%,with
halophilicActinopolysporaandNocardiopsisspeciesfromsalty a predicted minimum temperature of 23.3 to 29.2°C, an op-
VOL.62,1998 BIOLOGY OF MODERATELY HALOPHILIC AEROBIC BACTERIA 513
timumat20.0to23.4°C,andamaximumgrowthtemperature (v)Saltedfish,meat,andotherfoods.Althoughmoderately
of29.7to32.3°C(74)(theobservedminimumtemperaturewas halophilic bacteria are often found on salted fish, meat, and
25.4°C) (196); Halomonas meridiana, with two varieties: bio- other food products and regardless of their possible involve-
var I grows optimally at 1 to 3% NaCl and 28 to 40°C and mentinmicrobialspoilageathighsaltconcentrations,system-
toleratesNaClupto20to25%andtemperaturesupto45°C, aticstudiesontheoccurrenceofsuchbacteriaonfishandmeat
while biovar II grows best at 0.5 to 3% NaCl and 34 to 38°C productshaverarelybeenperformed.
andtoleratesupto25to30%NaCland47°Cbutisalsoable Moderate halophiles can easily be isolated from materials
togrowat25°C(134);andtwoyellow-orangeisolates,named suchassaltedanddriedfish,fishintestines,soysaucemashes,
FlavobacteriumgondwanenseandF.salegens,bothshowingop- andothersimilarmaterials(235,307).Recently,128strainsof
timalgrowthat5%NaClandgrowingatupto15to20%NaCl moderatehalophileswereisolatedfrombachalao(driedsalted
(52,55). codfish)andfromfreshcodandcuringsaltusedinitspreser-
The bacteria of Antarctic saline environments display an vation. In fully cured wet and dry bachalao (which contains
ability to grow at reduced temperatures compared with their about 19% salt), between 103 and 107 moderate halophiles
taxonomiccounterpartsfromtropicalandtemperateenviron- werefoundperg.Twoprimarycolonytypes,smoothandrough,
ments(195).Itwaspostulatedthatsincethesalinelakesofthe dominatedinwetanddrybachalao,respectively.Thebacteria
Vestfold Hills are geologically young, the resident bacteria forming the smooth colony type are similar to Halomonas
have not yet evolved mechanisms that may enable them to salinaandgrewin0.1to4.5MNaClat15to37°C(348).Inthe
grow in these environments in winter when temperatures fall curing of anchovies, Pediococcus halophilus became the dom-
belowtheirminimumrequiredforgrowthandthat,givenfur- inantbacteriumattheendofthecuringprocess;thisorganism
therevolution,organismsthatcanexploittheseenvironments developsunderbothaerobicandanaerobicconditions.Itshowed
morefullymaydevelop(73). optimum growth at 6.5 to 10% NaCl and tolerated over 15%
Also, the soils of the Antarctic Dry Valleys have yielded NaCl(349).
some interesting salt-tolerant bacteria, such as a halotolerant (vi) Unusual habitats. Moderately halophilic bacteria may
Planococcusthatgrowsfrom0to40°Cinthepresenceof0to be found in some unusual environments, such as on desert
2 M NaCl (207). Additional halotolerant gram-positive cocci plantsanddesertanimals.
wereobtainedfromsalinesoilsingeothermalregionsinAnt- Atriplex halinus (family Chenopodiaceae) is a desert plant
arctica. One such isolate, tentatively assigned to the genus widespread in the Negev Desert, Israel, and in other desert
Micrococcus,toleratesNaClconcentrationsbetween0and4.2 environments.Theleavesexcretesaltthroughsaltglands,the
M(221).Sincethisstrainoriginatedfromathermalareawith number and size of which depend on the amount of salt
in situ temperatures of up to 40°C, it is not surprising that it presentduringgrowthoftheplant.Salt(predominantlyNaCl)
doesnotsharethepsychrophilicorpsychrotolerantproperties crystallizes on the leaves when the salt gland bladders burst.
oftheotherstrainsmentionedabove:itsoptimumtemperature Duringthedryseason,significantamountsofsaltsandorganic
is37°C,andnogrowthwasobservedbelow20°C. material coat the leaf surface. The nightly occurrence of dew
(iv)Alkalinesalinehabitats.Stablealkalinehypersalineen- causes a diurnal wetting, so that the phylloplane microorgan-
vironments are not common and are the result of an unusual ismsexperiencelargefluctuationsinsalinityandwateractivity,
combination of geological, geographical, and climatic condi- includingrepeateddesiccation.Between13104and53105
tions (102). Most studies on the alkaline saline environments bacteria were enumerated per cm2 of leaf surface. The diver-
haveconcentratedonlakessuchasLakeMagadi,Kenya,and sity of culturable bacteria was limited, with the dominant or-
the Wadi Natrun lakes, Egypt, which are dominated by ex- ganism being an orange pigmented bacterium, identified as a
tremelyhalophilicarchaea. Pseudomonas sp., growing from 0.05% to 20% NaCl with an
AnovelrepresentativeoftheHalomonasgroupwasrecently optimumat5%and30°C(311).
isolated from sand of Venere Lake, Pantelleria Island, Italy. EvenmoreunusualistheisolationofahalotolerantBacillus
This aerobic pleomorphic rod, designated Halomonas pantel- sp. from the nasal cavities of desert iguanas. These animals
leriense, grows optimally at pH 9 (range, 7.5 to 11) and 10% possess salt glands in their nasal cavities that allow them to
NaCl(range,1.25to15%)(290).Someinterestinggram-neg- excrete a concentrated KCl brine during osmotic stress. A
ativehalophilicbacteriawereobtainedfromthealkaline(pH Bacillus sp. was isolated, showing excellent growth in 2.2 M
9.8),saline(9%totalsalts)MonoLake,Calif.Onestrainwas KClandwithanoptimumgrowthtemperatureof45°C(50).
isolatedonamediumcontainingglycinebetaineasthemajor
carbonandenergysource.Otherswereisolatedonamedium MorphologicalandPhysiologicalDiversity
containing dimethylsulfoniopropionate (DMSP). All isolates
grewatNaClconcentrationsfrom1.5to3M,andgrowthwas The preponderance of nonspecialized heterotrophs among
muchbetteratpH9.7thanatpH7.Glycinebetaineisdegrad- the known bacterial halophiles does not necessarily reflect
ed by sequential demethylation via dimethylglycine to sarco- their dominance in salt lakes, saline soils, and other saline
sine,whichisexcretedintothemedium.DMSPiseithercleaved habitatsbut,rather,maybeduetotherelativeeaseofcultur-
todimethylsulfideandacrylateordegradedbydemethylation, ing these bacteria (136). The halophilic property is probably
with 3-methiolpropionate as the intermediate, yielding meth- widespreadinthebacterialdomainandmayoccurinavariety
anethiol(32,51).GlycinebetaineandDMSPmaybefoundas of morphological and physiological types. This is nicely illus-
substratesforbacterialdegradationinMonoLake,sincethey tratedbythestudiesofHirsch,whodifferentiated104different
are produced by cyanobacteria and eukaryotic phytoplankton morphotypesofbacteriainthehypersalineSolarLakeonthe
asosmoticsolutes. shoreoftheSinaipeninsula.Thissmallandshallow(maximum
A haloalkaliphilic gram-positive bacterium was isolated from depth, about 5 m) thalassohaline lake is stratified in winter,
LakeGabaraintheWadiNatrun,Egypt,andnamedBacillus with an epilimnion containing 4.5 to 9% salt, increasing to
haloalkaliphilus;thisisolatetoleratesupto4MNaClbutgrows about 19% at the bottom. In addition to salt stress, tempera-
bestfrom0.5to3M(327,361).Additionalstrainsassignedto ture may be an important selective factor, since in the upper
the species B. haloalkaliphilus have been isolated from brine, hypolimnion heliothermal heating can increase the tempera-
driedsoil,mudanddungsamplesfromtheWadiNatrun(78). ture to about 60°C in winter. During the summer season, the