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FEMS Microbiology Reviews 39 (1986) 9-15
Published by Elsevier
9
FER 00021
The ecology and taxonomy of halobacteria
( Halobacterium," Halococcus; Natronobacterium; Natronococcus; saline brines; soda lakes;
halobacterial environments; archaebacterial halophiles)
W.D. Grant and H.N.M. Ross
Department of Microbiology, Universityof Leicester, LeicesterLEI 7RH, U.K.
Received 13 March 1986
Accepted 17 March 1986
1. SUMMARY
Archaebacterial halophiles dominate naturally
occurring brines as the concentration of salts approaches saturation. Although only 4 genera of
these bacteria have been recognised (Halobacterium, Halococcus, Natronobacterium, Natronococcus), chemotaxonomic studies indicate that
there are 4 or 5 additional distinct groups.
Archaebacterial halophiles are extremely physiologically versatile, some examples being capable of
an anaerobic fermentative mode of growth, others
of growth linked to the reduction of sulphur compounds. Many strains contain retinal-based pigments producing light-mediated movements of ions
across the cell membrane, that in some cases can
be harnessed for energy generation. However, it is
likely that under normal conditions, an aerobic,
chemoorganotrophic mode of nutrition is adopted.
2. INTRODUCTION
Archaebacterial halophiles (halobacteria) characteristically inhabit high salt environments such
as solar evaporation ponds (salterns) and naturally
occurring salt lakes, and can also be found on
products such as salted hides and fish, since solar
salt is traditionally used in the production of these
materials. These bacteria impart a red colouration
to these environments, due to the production of
C50 carotenoids (bacterioruberins). It was not until
the late 19th and early 20th century that the
microbiological nature of the reddening in salt
ponds and on salted products was realised [1] and
much later before the curious nature of these
organisms was subject to thorough biochemical
and physiological investigations [2]. Archaebacterial halophiles have in the main an obligate
requirement for at least 2 M NaC1 in the growth
medium and are thus extremely halophilic [1].
Although there are eubacteria categorised as extreme halophiles [3], the archaebacterial halophiles
may be unique in that it is not possible to obtain
mutants (or phenotypic variants) capable of growth
at low salt concentrations.
Until recently, only two major groups of these
bacteria were recognised, the rod-shaped or pleomorphic isolates (the genus Halobacterium) and
the coccoid isolates (the genus Halococcus). However, the discovery of the haloalkaliphilic archaebacteria of the genera Natronobacterium and
Natronococcus [4,5] has indicated that there may
be considerable diversity within the general
halophile phenotype, and a chemotaxonomic
survey of isolates from a variety of environments
has confirmed this view [6] indicating the presence
of 8 or 9 distinct groups.
0168-6445/86/$03.50 © 1986 Federation of European Microbiologicad Societies
10
CO~-
We review here the ecology of these remarkable
prokaryotes and discuss the need for a new taxonomy of the group.
Na÷
SO2-
S
3. T H E H A L O P H I L E E N V I R O N M E N T
The ion composition of a salt lake depends on
the surrounding topography, geology and the prevailing climatic conditions. Such lakes generally
develop in closed basins in tropical or sub-tropical
areas subject to constant high temperatures and
high light intensities. If evaporative concentration
exceeds the input of fresh waters, a highly saline
brine will develop. The genesis of such brines is
not completely understood, but it is clear that the
concentration of Ca 2+ and Mg 2+ (and to a lesser
extent SO42-) in the surrounding rocks is of
paramount importance [7].
Dilute solutions of anions and cations (usually
dominated by Na ÷, CI-, H C O 3 , CO32-) upon
concentration undergo precipitation events in the
presence of high amounts of Ca 2÷ (as CaCO 3) and
Mg 2+ (as Mg2Si308 • nH20); a further precipitation stage may occur in high-SO 2 - brines (as
CaSO4). The removal of these ions as essentially
insoluble salts clearly influences the final composition of the brines, but there is also an important effect on the final pH in that the precipitation of CO 2- removes alkalinity, and the formation of sepiolite (Mg2Si3Os) generates H r. Thus,
a high Ca 2 + area will tend to produce a neutral to
slightly alkaline mainly Na* + C1- brine (as in the
Great Salt Lake), a high Ca2+Mg 2+ area, a neutral
to slightly acid Mg 2+ + Na + + C1- brine (as in the
Dead Sea), and a low Mg2+Ca 2+ area an extremely alkaline Na ÷ + C1- + CO 2+ brine (e.g.,
Lake Magadi, Kenya) (Fig. 1).
As the total dissolved salts in such environments approaches saturation, halobacteria are able
to outcompete eubacteria and eukaryotes capable
of growth at high salt concentrations [8-11] and it
is rare to culture bacteria other than archaebacteria from these environments. Salt lakes are, in
general, highly productive, with considerable
amounts of dissolved organic carbon [8-10] and
the origins of this material are not entirely understood. In neutral salt lakes, eukaryotic algae of the
SiO2
UCa2÷
K÷
Mg2÷
J
Low Ca2÷
High Ca2+
so~-]
[ so~- I
[
HCO]
1
Low Mg2.
Calcite
CaCO3
precipitation
High Ca2÷
[
so~- ]
High Hg2+
Catcife
CaCO3
precipitation
Sepiolife
Hg2Si308 "nH20
precipitation
I
Gypsum ]
CaSO4
precipitation
[ oypsuo]
CaSO4
precipitation
l
Na*
CO~- Ct-
Na÷ C[-
Na+ Mg2÷ C[-
[SO 2-]
[SO 2-]
[ SO2-]
SODA LAKE
pH 10-11"
SALT LAKE
pH 7-8
SALT LAKE
pH 6-7
C~EAT SALT LAKE
DEAD SEA
LAKE MAGADI
Fig. 1. The effect of Ca 2+ and Mg 2+ on the genesis of highly
saline brines.
genus Dunaliella are significant primary producers
over a wide range of salt concentrations, but probably not under conditions where halobacteria
bloom [8,9]. In alkaline salt lakes, the position is
less clear; Dunaliella spp. have been described in
some of the lakes of the Egyptian Wadi Natrun
[10] but not in the Kenyan Lake Magadi [12]; it is
likely that in alkaline lakes primary productivity is
due to anoxygenic phototrophic bacteria of the
genus Ectothiorhodospira [10,12] growing under
much the same conditions of salt as Dunaliella
spp. but occasionally under anoxic conditions,
outcompeting halobacteria in highly saline environments [10,12]. At present, our knowledge of
11
microbial successions in these lakes is sparse and
largely deductive, but it would not be unreasonable to suppose primary production during periods
of dilution (say during a short rainy season), followed by organotrophic successions culminating
in a halobacterial bloom as dissolved salts approach saturation.
4. T A X O N O M Y A N D
HALOBACTERIA
PHYLOGENY
OF
Difficulties in assigning halobacterial isolates
to the appropriate taxa arise in that many isolates
are somewhat biochemically inert and thus phenotypically similar. However, the thorough taxonomic studies of Kocur and Hodgkiss [13] indicate
that all coccoid strains comprise a monospecific
genus with Halococcus morrhuae as the sole
species. On the other hand, the rod-shaped and
fractional stability
~8
if6
I
'
I
I
I
1-0
I
El4
q.J
eW
~cJ
I'-<C
o=
..I
[--
I-
-J
.I-
ua
j
Hatobacterium halobium
Halobacferium cufirubrum
Halobacterium salinarium
CCH 2090
CCH 2088
CCH 2168
fiENUS 1
Haiobacterium halobium
Halobacterium salinarium
Haiobacterium trapanicum
NCMB777
NCMB7116
NCMB 784
GENUS2
"HALONEHA"
Halobacferium volcanii
Halobacterium mediterranei
NCMB2012
CCM 3361
GENUS 3
"HALOSINU$"
Natronobacterium pharaonis
Natronobacterium mag~lli.
Nafronobacterium gregoryl
DSM 2160
NCMB2190
NCMB2189
GENUS4
NATROI~ACTERIUH
NatronococcUs occultus
NCMB2192
GENUSS
NATRONOCOCCUS
Halococcus morrhuae
Halococcus morrhuae
NCMB787
NCMB 761
GENUS6
HALOCOCCUS
Halobacterium vaUismorfi$
"Halobacferium
marismortui"
I
~c
pleomorphic isolates are, in general, phenotypically similar, and even quite extensive numerical
taxonomic analyses have proved less than useful
[14]. Attempts to reclassify certain pleomorphic
isolates as a new genus Haloarcula must be called
into question, in view of the similarity of the
isolates to certain other halobacteria [15] and the
general ubiquity of disc-shaped or rectangular isolates.
The discovery of the haloalkaliphile phenotypes
[4,5] (high pH requirement, low Mg 2÷ requirement) from soda lakes prompted a reappraisal of
the group as a whole on the basis of chemotaxonomic markers rather than standard biochemical
tests [6,16]. Polar lipid analyses, D N A / D N A
base-pairing studies and 16s R N A / D N A hybridisation studies, indicate that there are at least
5 other major groups in addition to the validly
published genera Halococcus, Halobacterium,
Natronobacterium and Natronococcus. These re-
I
HALOBACTERIUM
ATCC 29715 GENUS 7
"HALOPI.ANUS"
Halobacferium $accharovorum
Ha[obacferiumfrapanicum
Halobacferium SOClomense
NCMB2081
NRC 34021 GENUSB
ATCC 33755
"HALOI~UIBRLMq
'°
Hatobacterium cufirubrum
N C M B 7 6 3 GENUS9
"HALOTHRIX"
Fig. 2. Relationships amongst halobacteriabased on the thermal stability of 16 S rRNA/DNA hybrids (modified from [6]). ATCC,
American Type Culture Collection; CCM, Czechoslovak Collection of Microorganisms; DSM, Deutsche Sammlung yon
Mikroorganismen; NCMB, National Collectionof Marine Bacteria, U.K. 'Halobacterium marismortui' resembles the lost type strain
and was kindly supplied by Dr. M. Ginzburg. Names in quotes are not validlypublished.
12
sults agree well with groups obtained by 5 S
rRNA sequence comparisons (G.E. Fox, personal
communication). Fig. 2 shows relationships based
on the stability of D N A / 1 6 S rRNA hybrids and
indicates how a new taxonomy might be constructed with two new families defined by the
original Halobacterium and Halococcus genera.
D N A / D N A base pairings indicate a total of 17
potential species within these putative generic
groupings, although the type strains of Halobacterium salinariurrg Halobacterium halobium and
Halobacterium cutirubrum are related at > 70%
and should be considered as different isolates of
the same species (Hb. salinarium has historical
precedence) [6]. It will be noted that there are a
number of largely unrelated isolates with identical
specific epithets.
Table 1 lists the origins of the strains used in
this study and indicates that in some cases all the
organisms from a group are derived from similar
environments, but there are many exceptions,
where organisms grouped together derive from
both different saline environments and widely different geographical locations. It is also worth noting that virtually all the biochemical studies on
halobacteria have been carried out with organisms
from Genus 1, a biochemically unreactive group.
A new taxonomy for these archaebacteria comparable to those taxonomies accepted for other
archaebacteria is long overdue. Ross [17] has detailed proposals for such.
5. H A L O B A C T E R I A AS A R C H A E B A C T E R I A
Long before the archaebacteria were defined by
Woese and colleagues [18], there was a clear perception that the halobacteria constituted an unusual group of prokaryotes. In particular the classic
liPid work by Kates et al. [19] established the
presence of ether-linked isopranoid lipids as the
main membrane structural components, one of the
Table 1
Origins of halobacterial isolates
Genus I
Halobacterium halobium CCM2090 a
Halobacterium cutirubrum CCM2088
Halobacterium salinarium CCM2148
Salted fish
Salted fish
Salted fish
Genus 2
Halobacterium halobium NCMB777
Halobacterium salinarium NCMB786
Halobacteriurn trapanicum NCMB784
Salted hides
Salted hides
Salted hides
Genus 3
Halobacteriurn volcanii NCMB2012
Halobacterium mediterranei CCM3361
Dead Sea
Saltern, Spain
Genus 4
Natronobacterium pharaonis DSM2160
Natronobacteriurn magadii NCMB2190
Natronobacterium gregoryi NCMB2189
Wadi Natrun, Egypt
Lake Magadi, Kenya
Lake Magadi, Kenya
Genus 5
Natronococcus occultus NCMB2192
Lake Magadi, Kenya
Genus 6
Halococcus morrhuae NCMB787
Halococcus morrhuae NCMB761
Salted fish
Salted fish
Genus 7
Halobacterium oallismortis ATCC29715
"Halobacterium marisrnortui" M. Ginzburg
Salt Lake, California
Dead Sea
Genus 8
Halobacterium saecharovorurn NCMB2081
Halobacterium trapanicum NRC34021
Halobacterium sodomense ATCC33735
Saltern, California
Saltern, Italy
Dead Sea
Genus 9
Halobacterium cutirubrum NCMB763
Salted fish
a Abbreviations as in Fig. 2.
13
key features of archaebacteria as presently defined. It is now clear, of course, that halobacteria,
in common with methanogenic and thermophilic
and sulphur-utilising archaebacteria also show
characteristic inhibitor sensitivities, lack peptidoglycan-containing cell walls, have characteristic
RNA polymerases and so on [see 20] relating to
the general archaebacterial phenotype. However,
halobacteria seem to lack the C40, C40 dibiphtanyl
diglycerol tetraethers characteristic of the other
groups of archaebacteria, their membranes being
composed in the main of C20, C20 diphytanyl
glycerol diethers [21,22]. However, certain groups,
notably the haloalkaliphiles possess substantial
amounts of C25 (sesterterpanyl) chains in ether
linkage to glycerol as 2-O-sesterterpanyl-3-Ophytanyl diether, and rarely as di-sesterterpanyl
diether (C25, C25) [23,24] (Fig. 3).
Special halophile features include the posses-
!
H
H
2 - O ~
~ O ~
0.t2-0tt
cl
H2-O~
H
CH2-OH
b
I
-
O
~
CH2-OH
c
Fig. 3. Core glycerol ether lipids of halobacteria. (a) Diphytanylglycerol diether (C2o,C2o); (b) 2-O-sesterterpanyl-3O-phytanylglycerol ether (C20,C25); (c) disesterterpanyl
glycerol diether (Ce5,C25).
sion of retinal-based pigments capable of producing movements of ions across the cell membrane
[25], and a characteristic cell wall structural
eukaryote-like glycoprotein requiring high concentrations of Na + ions for structural integrity,
found in all but one of the groups shown in Fig. 2
([26], Stevens and Grant, unpublished results).
There are 3 archaebacterial retinal-based pigments known which undergo light-mediated cycling of energised states all with absorption maxima between 560-590 nm, an early short-lived
red-shift intermediate and a longer-lived intermediate absorbing between 370 and 420 nm, the 3
pigments differing from one another in the rate of
interconversion of these energised states. The first
pigment to be described, bacteriorhodopsin, is an
outwardly directed proton pump and the proton
gradient so produced can be harnessed for photophosphorylation. The second form to be discovered, halorhodopsin, is an inwardly directed
chloride pump, and thus presumably involved in
cell ion homeostasis. The third, slow rhodopsin,
appears to be a transducer in the phototactic
response of halobacteria. These light-mediated ion
movements are unique to halophilic archaebacteria in the prokaryote realm, and include the
only known example of a non-chlorophyll-mediated photophosphorylation mechanism. However,
it is worth noting again that all the detailed studies of retinal pigments (and structural glycoproteins) have been carried out on organisms from
Genus 1. There is no guarantee that the retinal
systems are common to all halobacteria, since
there is evidence that bacteriorhodopsin in particular is absent in some groups, notably the
haloalkaliphiles (Stoeckenius, personal communication).
The relationship of halobacteria to other
archaebacteria (as deduced by DNA or RNA sequence comparisons) is the subject of some dispute. There is general agreement that halophiles
are most closely related to the methanogens, and
early comparisons of 16S rRNA catalogues suggested a relationship with the Methanobacteriales
[27]. However, recent more detailed comparisons
of complete 16 S rRNA gene sequences have
indicated a more close relationship with the
Methanomicrobiales [28].
14
6. H A L O B A C T E R I A
ENVIRONMENT
IN
THE
NATURAL
Halobacteria are traditionally cultured aerobically on high-nutrient media, i.e., as chemoorganotrophs. However, in view of the report that
certain strains are capable of an anaerobic
fermentative mode of growth [29], and the observation that an anaplerotic type of CO 2 fixation
occurs during retinal-mediated photophosphorylation [30] under anaerobic conditions, it is pertinent to ask what kind of physiology these bacteria
exhibit in the natural environment. Very few field
studies have been carried out, but the results
obtained by Oren [31] in a systematic study of the
Dead Sea give some indications of halobacterial
activities in this high Mg 2+ brine. Using inhibitors
of eukaryotic photosynthesis (to inhibit Dunaliella
cells in the main) and light of different wavelengths (to detect bacteriorhodopsin-mediated activity) it is possible to discriminate between
primary productivity due to Dunaliella cells (and
other eukaryotes) and that due to halobacteria.
Halobacterial light-dependent CO 2 fixation is
observed under microaerophilic conditions (the
norm in highly saline waters), but the amount of
CO 2 fixed per unit of halobacterial biomass is at
least one or two orders of magnitude less than that
fixed by an equivalent biomass of Dunaliella ceils.
These results suggest that halobacteria probably
do not grow primarily as photolithotrophs, relying
on bacteriorhodopsin-mediated photophosphorylation (and concomitant CO 2 fixation) under less
than ideal conditions (i.e., temporary anaerobiosis). There is no doubt that bacteriorhodopsin has
a protective effect on loss of viability in anaerobic
starved cells in the light [32]. The fermentative
mode of metabolism exhibited by some strains
[29] may be another example of a contingency
mechanism.
There is one further possibility open to cells
under anaerobic conditions that has yet to be
examined in detail, in that m a n y strains that we
have examined are capable of anaerobic growth
coupled to the reduction of sulphur compounds
(elemental S, S203z- but probably not SO 2 - ) . This
last attribute appears to be a general archaebacterial characteristic since the sulphur-depen-
dent archaebacteria possess this attribute by definition, and methanogens have been shown to be
facultative reducers of sulphur compounds [33].
7. C O N C L U S I O N S
Halophilic archaebacteria constitute a remarkably versatile group of prokaryotes, with a variety
of nutrition and energy options open to cells
under adverse conditions.
It is clear that there is considerable genetic
diversity amongst existing isolates of halobacteria,
and since the majority of these have been isolated
as aerobic organotrophs on similar complex media,
the use of different isolation procedures should
yield even more diverse types - - Walsby's square
bacterium, for example, although almost certainly
a halophilic archaebacterium [34,35], has yet to be
isolated.
The widely held belief that archaebacterial
halophiles could only grow aerobically has been
advanced as supportive evidence for the view that
these bacteria evolved later than other archaebacteria, i.e., after the appearance of oxygen in the
Earth's early atmosphere. Clearly it is no longer
possible to argue this case in this particular way.
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