micb-13-799362 Ma ch 11, 2022 Time: 16:49 # 1
ORIGINAL RESEARCH
published: 17 Ma ch 2022
doi: 10.3389/ micb.2022.799362
Edi ed by:
Takema Fuka su,
Na ional Ins i u e o Ad anced
Indus ial Science and Technology
(AIST), Japan
Re iewed by:
Te esa Thiel,
Uni e si y o Missou i–S . Louis,
Uni ed S a es
I ina N. Shilo a,
Second Genome, Uni ed S a es
*Co espondence:
En ique Flo es
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Mic obial Symbioses,
a sec ion o he jou nal
F on ie s in Mic obiology
Recei ed: 21 Oc obe 2021
Accep ed: 22 Feb ua y 2022
Published: 17 Ma ch 2022
Ci a ion:
Flo es E, Romano icz DK,
Nie es-Mo ión M, Fos e RA and
Villa eal TA (2022) Adap a ion o an
In acellula Li es yle by
a Ni ogen-Fixing,
He e ocys -Fo ming Cyanobac e ial
Endosymbion o a Dia om.
F on . Mic obiol. 13:799362.
doi: 10.3389/ micb.2022.799362
Adap a ion o an In acellula
Li es yle by a Ni ogen-Fixing,
He e ocys -Fo ming Cyanobac e ial
Endosymbion o a Dia om
En ique Flo es1*, Dwigh K. Romano icz2, Me cedes Nie es-Mo ión1, Rachel A. Fos e 3
and T acy A. Villa eal4
1Ins i u o de Bioquímica Vege al y Fo osín esis, CSIC, Uni e sidad de Se illa, Se ille, Spain, 2Depa men o Psychology,
The Uni e si y o Texas a Aus in, Aus in, TX, Uni ed S a es, 3Depa men o Ecology, En i onmen and Plan Sciences,
S ockholm Uni e si y, S ockholm, Sweden, 4Depa men o Ma ine Science and Ma ine Science Ins i u e, The Uni e si y
o Texas a Aus in, Po A ansas, TX, Uni ed S a es
The symbiosis be ween he dia om Hemiaulus hauckii and he he e ocys - o ming
cyanobac e ium Richelia in acellula is makes an impo an con ibu ion o new
p oduc ion in he wo ld’s oceans, bu i s s udy is limi ed by sho - e m su i al in
he labo a o y. In his symbiosis, R. in acellula is ixes a mosphe ic dini ogen in he
he e ocys and p o ides H. hauckii wi h ixed ni ogen. He e, we conduc ed an elec on
mic oscopy s udy o H. hauckii and ound ha he ilamen s o he R. in acellula is
symbion , ypically composed o one e minal he e ocys and h ee o ou ege a i e
cells, a e loca ed in he dia om’s cy oplasm no enclosed by a hos memb ane. A second
p oka yo ic cell was also de ec ed in he cy oplasm o H. hauckii, bu obse a ions we e
in equen . The he e ocys s o R. in acellula is di e om hose o ee-li ing he e ocys -
o ming cyanobac e ia in ha he speci ic componen s o he he e ocys en elope seem
o be loca ed in he pe iplasmic space ins ead o ou side he ou e memb ane. This
specialized a angemen o he he e ocys en elope and a possible associa ion o he
cyanobac e ium wi h oxygen- espi ing mi ochond ia may be impo an o p o ec ion o
he ni ogen- ixing enzyme, ni ogenase, om pho osyn he ically p oduced oxygen. The
cell en elope o he ege a i e cells o R. in acellula is con ained nume ous memb ane
esicles ha esemble he ou e -inne memb ane esicles o G am-nega i e bac e ia.
These esicles can expo cy oplasmic ma e ial om he bac e ial cell and, he e o e,
may ep esen a ehicle o ans e o ixed ni ogen om R. in acellula is o he
dia om’s cy oplasm. The speci ic mo phological ea u es o R. in acellula is desc ibed
he e, oge he wi h i s known s eamlined genome, likely ep esen speci ic adap a ions
o his cyanobac e ium o an in acellula li es yle.
Keywo ds: cyanobac e ia, dia om, Hemiaulus hauckii, he e ocys , memb ane esicles, Richelia in acellula is,
symbiosis
F on ie s in Mic obiology | www. on ie sin.o g 1Ma ch 2022 | Volume 13 | A icle 799362
micb-13-799362 Ma ch 11, 2022 Time: 16:49 # 2
Flo es e al. Dia om-Cyanobac e ium Ni ogen-Fixing Symbiosis
INTRODUCTION
Dia oms (Bacilla iophyceae) a e a mo phologically and
ecologically di e se g oup o algae ha ha e a cell wall, known as
a “ us ule,” ha inco po a es silica as a main componen (Round
e al., 2007). The us ule consis s o wo hal es ( al es), he
smalle hypo heca and he la ge epi heca, ha i one in o he
o he and can show adial o bila e al symme y (Round e al.,
2007). Al hough dia oms a e equen ly ound as single cells,
some can o m chains o cells as in he case o Hemiaulus hauckii
(Pyle e al., 2020). As in o he euka yo es, he ul as uc u e o
a dia om cell con ains a nucleus, Golgi complex and abundan
mi ochond ia, bu also chlo oplas s (Bedosh ili and Likhoshway,
2012). Dia oms a e dis ibu ed wo ldwide, and in oceanic
low-nu ien a eas dia om-diazo ophic associa ions p oli e a e
widely (Mague e al., 1974;Ven ick, 1974;Villa eal e al., 2012;
Ande son e al., 2018;Pie ella Ka lusich e al., 2021), making an
impo an con ibu ion o CO2 ixa ion (p ima y p oduc i i y)
suppo ed by N2 ixa ion (diazo ophy) (Sub amaniam e al.,
2008;Ka l e al., 2012). Some o hese associa ions in ol e
a ilamen ous, he e ocys - o ming cyanobac e ium, Richelia
in acellula is, as he N2- ixing pa ne (Taylo , 1982;Villa eal,
1992).
Cyanobac e ia ca y ou oxygenic pho osyn hesis, and because
he N2- ixing enzyme, ni ogenase, is sensi i e o oxygen, hese
o ganisms usually sepa a e ni ogen ixa ion om pho osyn hesis
in ei he ime o space (Flo es e al., 2015). In he e ocys - o ming
cyanobac e ia, N2 ixa ion unde oxic condi ions is con ined
o he e ocys s, which a e mo phologically- and me abolically-
di e en ia ed cells sui ed o N2 ixa ion (Kuma e al., 2010).
He e ocys s p o ide he ege a i e cells o he ilamen wi h
ixed ni ogen, and he ege a i e cells p o ide he he e ocys s
wi h educed ca bon ha is needed o uel N2 ixa ion (He e o
e al., 2016). Richelia sp. belongs o he o de Nos ocales and,
simila o young ichomes o Calo h ix sp. and Scy onema
sp. (Rippka e al., 1979), o ms sho ilamen s wi h e minal
he e ocys s (Villa eal, 1991;Fos e e al., 2011;Hil on e al.,
2013). Richelia in acellula is can be ound associa ed wi h
dia oms such as Hemiaulus hauckii,H. memb anaceus and
Rhizosolenia cle ei. In he la e , he cyanobac e ium is ound
in a “pe iplasmic” loca ion, i.e., si ua ed be ween he dia om’s
cy oplasmic memb ane and us ule (Villa eal, 1990;Janson e al.,
1995), whe eas in H. hauckii he cyanobac e ium appea s o be
loca ed in he cy oplasm (Capu o e al., 2019). The d a genomes
o Richelia symbion s o H. hauckii and R. cle ei di e , whe e he
in e nal Richelia genome is a educed con aining incomple e
sequences and lacking, o example, se e al N assimila o y
pa hways (Hil on e al., 2013;Nie es-Mo ión e al., 2020).
In acellula bac e ia in euka yo ic cells can be ound su ounded
by a memb ane o he hos (as in a memb ane-bound acuole)
o esiding di ec ly in he cy oplasm (Salje, 2021), bu how
in eg a ed he R. in acellula is is o he cy oplasm o H. hauckii
is unknown.
The cyanobac e ia ha e a G am s ain-nega i e ype o cell
en elope in which a cy oplasmic memb ane, a pep idoglycan
laye and an ou e memb ane can be disce ned (Hahn and
Schlei , 2014). The he e ocys s o ee-li ing he e ocys - o ming
cyanobac e ia, including he model s ain Anabaena sp. PCC
7120 (he ea e Anabaena), bea ou side o he ou e memb ane
an ex a en elope ha is composed o an inne lamina ed
glycolipid laye (Hgl) and an ou e homogeneous polysaccha ide
laye (Hep) (Lang and Fay, 1971;Nie zwicki-Baue e al., 1984;
Adams and Duggan, 1999;Flo es e al., 2006). The Hgl laye
appea s o es ic oxygen di usion in o he he e ocys whe eas
he Hep laye seems o p o ec he Hgl laye om dispe sal
in o he medium (Xu e al., 2008;Nicolaisen e al., 2009).
In e es ingly, Hemiaulus-associa ed Richelia p oduce dis inc
glycolipids ha con ain ibose ins ead o he hexose and longe
aglycone moie ies han hose ound in ee-li ing he e ocys -
o ming cyanobac e ia (Schou en e al., 2013;Bale e al., 2015).
In he he e ocys s o Anabaena sp., he in acellula memb anes
cha ac e is ic o cyanobac e ia ( he hylakoids ha ca y ou
pho osyn hesis) a e concen a ed close o he he e ocys poles,
whe e hey o m a “honeycomb”-like s uc u e (Lang and Fay,
1971). Te minal espi a o y oxidases, which can consume oxygen
ha en e s he he e ocys h ough he he e ocys - ege a i e cell
junc ions, appea o be loca ed a hese honeycomb memb anes
(Mu y e al., 1981;Vallada es e al., 2007;To ado e al.,
2019). He e ocys s can be ound in e cala y o e minally in
he Anabaena ilamen s. A he he e ocys pole nex o a
ege a i e cell, he he e ocys p esen s a na owing ha o ms
he so called “he e ocys neck” (Flo es and He e o, 2010).
Cyanophycin, a ni ogen ese oi composed o aspa a e and
a ginine, accumula es wi hin he he e ocys neck and nex o i
(She man e al., 2000).
In his wo k, we p esen an elec on mic oscopic s udy
o H. hauckii wi h an emphasis on i s endosymbion ,
R. in acellula is. We show ha R. in acellula is esides
di ec ly in he cy oplasm o he dia om. Addi ionally, a
second p oka yo ic o ganism, which was less equen ly
obse ed, appea ed also in eg a ed in he H. hauckii cy oplasm.
The cell en elope o R. in acellula is con ains nume ous
memb ane esicles and shows a close associa ion o he
dia om hos ’s mi ochond ia. We also ound ha he e ocys
mo phology is di e en in R. in acellula is han in ee-li ing
he e ocys - o ming cyanobac e ia.
MATERIALS AND METHODS
This s udy was pe o med wi h symbio ic Hemiaulus hauckii cells
g own om samples collec ed wi h ne ows in he channel a Po
A ansas, Texas, Gul o Mexico in Fall 2012. La e exponen ial
phase, dense cul u es we e p epa ed using an a i icial seawa e
medium as desc ibed in Pyle e al. (2020). Cul u es we e g own
a 25◦C on a 12-h ligh :12-h da k cycle. F om hese cul u es,
indi idual sho chains o dia om cells con aining symbion s we e
isola ed by mic opipe e unde a s e eomic oscope and pelle ed
o a ached o a luo oca bon subs a e (ACLAR) wi h poly-L-Lys
o subsequen p ocessing.
Fo elec on mic oscopy, cells we e ini ially ixed o e nigh
a oom empe a u e, in a mix u e o EM-g ade aldehyde
ixa i es: 4% glu a aldehyde and 2% o maldehyde in 0.1 M
cacodyla e bu e a pH 7.4 wi h 35 mg/mL Ins an Ocean.
A e inses in bu e wi h dec easing salini y, he cells we e
ixed in educed osmium (a mix u e o 2% osmium e oxide
F on ie s in Mic obiology | www. on ie sin.o g 2Ma ch 2022 | Volume 13 | A icle 799362
micb-13-799362 Ma ch 11, 2022 Time: 16:49 # 3
Flo es e al. Dia om-Cyanobac e ium Ni ogen-Fixing Symbiosis
and 2% po assium e ocyanide in 0.1 M cacodyla e bu e , pH
7.4). Be o e addi ional p ocessing, he cell p epa a ions we e
dehyd a ed in an e hanol se ies, in which 2% u anyl ace a e
was inco po a ed in he 70% e hanol s ep. F om his poin
he e we e wo pa hs: (i) cells o scanning elec on mic oscopy
(SEM) we e dehyd a ed wi h c i ical poin d ying (CPD) and
hen coa ed wi h 15 nm o pla inum-palladium and imaged wi h
a Zeiss Sup a 40VP SEM; (ii) cells o ansmission elec on
mic oscopy (TEM) we e embedded in Epon Ha d 812 (Elec on
Mic oscopy Sciences)1, om which 70-nm sec ions we e picked
up on Fo m a -coa ed g ids and imaged wi h a Tecnai T12 TEM,
ope a ed a 80 kV.
Measu emen s o cell cha ac e s we e made in ImageJ
(Schneide e al., 2012). B ie ly, selec images we e impo ed in o
ImageJ, he embedded scale ba om he mic og aph was used
o calib a e he scale using he line ool, and h ee consecu i e
linea measu emen s we e made o he ollowing cell cha ac e s:
he e ocys and ege a i e cell dimensions (ci cula c oss-sec ion
and cell leng h), us ule and spine dimensions (diame e and
leng h), and dis ance be ween cells o a chain o H. hauckii.
The a e age and s anda d de ia ion o he h ee measu emen s
is epo ed. Addi ionally, he numbe s o chlo oplas s pe
H. hauckii we e also enume a ed when c oss sec ioning allowed
ull isualiza ion.
The sea ch o speci ic genes in he genome sequence o
R. in acellula is s ain HH01 (a ailable a h ps://img.jgi.doe.
go /cgi-bin/m/main.cgi) was pe o med by BLASTp analysis
(Al schul e al., 1997) using p o eins om Anabaena sp. s ain
PCC 7120 as que ies.
RESULTS
Chains o Hemiaulus hauckii Cells
A sample o H. hauckii isualized by scanning elec on
mic oscopy (SEM) showed chains o dia om cells (Figu e 1).
In hese chains, he cells a e joined by ele a ions wi h
e minal spines (Hasle and Sy e sen, 1996) o he us ule;
he leng h (15.6 ±3.7 µm) and wid h (1.35 ±0.3 µm) o
he ele a ions a ied nea ly wo- old in combined obse a ions
(Figu e 1 and Table 1). The dis ance be ween he cells a ied
om 5 o 12.5 µm. The cells in his sample we e abou
20 µm wide and 44–82 µm long (Table 1). The longes cells
as iewed by scanning mic oscopy can indeed be us ules
con aining wo cells, each o which con ains a endosymbio ic
cyanobac e ium, R. in acellula is (Figu e 1B). The us ule was
obse ed o con ain one la ge po e on i s su ace (Figu e 1A,
inse and Supplemen a y Figu es 1A,B; po e wid h, abou
200 nm) and nume ous small po es in i s in e nal ace
(Supplemen a y Figu es 1C,D). Dia oms possess di e en ypes
o po es wi h complex s uc u es and unc ion, including he
labia e p ocess and po es ha can allow nu ien and was e
di usion h ough he us ules (Medlin e al., 1986;De Tommasi
e al., 2017).
1h ps://www.emsdiasum.com
FIGURE 1 | Chains o Hemiaulus hauckii cells. (A) Scanning elec on
mic og aph o a chain o cells ha a e joined by hin pa allel ex ensions
(ele a ions) o he us ule. Inse , magni ied iew o ele a ions; he ci cle
highligh s he possible ex e nal opening o he labia e p ocess. (B) O e lay o
ligh and epi luo escence mic og aphs (exci a ion il e a 490 nm; emission,
565 nm) o pa o a chain o H. hauckii cells (wa e moun ) illus a ing ha he
longe us ules in panel A can con ain wo dia om cells each including
endosymbio ic Richelia in acellula is ( ilamen s o cells wi h yellow-o ange
luo escence).
Hemiaulus hauckii Cellula S uc u es
Samples o H. hauckii we e p epa ed o ansmission elec on
mic oscopy (TEM). Longi udinal (Figu es 2A,B) and ans e se
(Figu es 2C,D) sec ions o single dia om cells o pai s o
cells we e equen ly obse ed. As expec ed, he dia oms
we e su ounded by hei us ule, and in some mic og aphs
in e nal s uc u es such as he nucleus (including he nucleolus),
mi ochond ia, Golgi bodies, and nume ous chlo oplas s o
chlo oplas lobes (8 ±2 pe H. hauckii, n = 20; Figu e 2C and
Supplemen a y Figu e 2) we e e iden . A de eloping al e can
be obse ed in he lowe -le pa o he dia om cell in Figu e 2D,
indica ing ha i is a cell in he p ocess o di ision. Magni ica ion
o an a ea including he in e nal al e showed ha i is wi hin
a memb ane esicle (Supplemen a y Figu es 3A,B), sugges ing
ha i is indeed a ecen ly syn hesized al e ha is s ill wi hin
he silicalemma o silica-deposi ion esicle (C aw o d, 1981;
Hildeb and e al., 2018). A magni ied iew o an a ea including
chlo oplas s showed he p esence o plas oglobules (lipid
d ople s) in one o hem (Supplemen a y Figu e 2B). O he
han he dia om s uc u es, cyanobac e ial cells o ilamen s
we e obse ed in mos dia om cells (e.g., Figu es 2A,C,D and
Supplemen a y Figu e 2A), and we did no gene ally no ice he
p esence o o he bac e ial endosymbion s (see, howe e , below).
Richelia in acellula is Resides in he
Hemiaulus hauckii Cy oplasm
We nex ocused on he cyanobac e ia ha could be obse ed
wi hin he dia om cells. As shown in Figu e 1B,H. hauckii
con ains ilamen s o he he e ocys - o ming cyanobac e ium
R. in acellula is. Depending on he o ien a ion o he specimen,
F on ie s in Mic obiology | www. on ie sin.o g 3Ma ch 2022 | Volume 13 | A icle 799362
micb-13-799362 Ma ch 11, 2022 Time: 16:49 # 4
Flo es e al. Dia om-Cyanobac e ium Ni ogen-Fixing Symbiosis
TABLE 1 | Summa y o cell measu emen s om scanning elec on mic oscopy (SEM) and ansmission elec on mic oscopy (TEM) mic og aphs o Hemiaulus
hauckii-Richelia in acellula is symbioses.
Cell cha ac e Numbe o obse a ions A e age diame e ±SD
(min–max), µm
A e age leng h ±SD
(min-max), µm
Hemiaulus hauckii us ule 8 20.6 ±0.98
(19.1–21.8)
62.3 ±16.4
(44.2–82.1)
Cell–cell linking ele a ion 6 1.35 ±0.3
(0.92–1.7)
15.6 ±3.7
(12.7–22.7)
Richelia he e ocys 8 3.97 ±0.74
(2.65–4.90)
3.42 ±0.91
(2.36–4.82)
Richelia ege a i e cells 69 2.62 ±0.85
(0.67–4.41)
2.13 ±0.87
(0.42–3.95)
Full Richelia ilamen s 6
Vege a i e cell 1 1.96 ±0.26
(1.53–2.21)
2.44 ±0.4
(1.99–3.0)
Vege a i e cell 2 1.67 ±0.27
(1.35–1.97)
2.21 ±0.36
(1.82–2.75)
Vege a i e cell 3 1.52 ±0.50
(0.78–2.07)
2.06 ±0.58
(1.26–3.03)
Vege a i e cell 4 1.55 ±0.59
(0.69–2.38)
1.63 ±0.54
(0.92–2.29)
Full Richelia ilamen s de ined as ou ege a i e cells and one e minal he e ocys ; cell 1–4, close o a he o/ om he he e ocys . SD, s anda d de ia ion.
FIGURE 2 | T ansmission elec on mic og aphs o H. hauckii. Longi udinal sec ions o a single cell (A) o o a pai o cells (B) and ans e se sec ions o single cells
(C,D) can be obse ed. Filamen s o R. in acellula is (Rin ) can be seen in panels (A,D) (He , he e ocys ), and a ans e se sec ion o a cyanobac e ial cell (CB) can
be seen in (C) ( his cell is likely a ege a i e cell o a R. in acellula is ilamen ). Addi ionally, some dia om s uc u es a e e iden : us ules ( ) can be seen as he
ou e mos laye s in he cells o he ou mic og aphs, a de eloping al e can be seen in he lowe le pa o he cell in panel (D) (d ), and he nucleus (N), including
he nucleolus (nu), and se e al chlo oplas lobes (Ch) a e e iden in he dia om cell in panel (C).
F on ie s in Mic obiology | www. on ie sin.o g 4Ma ch 2022 | Volume 13 | A icle 799362
micb-13-799362 Ma ch 11, 2022 Time: 16:49 # 5
Flo es e al. Dia om-Cyanobac e ium Ni ogen-Fixing Symbiosis
FIGURE 3 | T ansmission elec on mic og aph o a H. hauckii cell.
(A) T ans e se sec ion o he dia om con aining wo cyanobac e ial cells (CB)
also mainly in ans e se sec ion; , dia om us ule. (B) Magni ied iew o he
dia om cy oplasm and pa o he cyanobac e ium indica ed by he do ed
squa e in panel (A). No e componen s o he cyanobac e ial cell en elope:
CM, cy oplasmic memb ane; PG, pep idoglycan laye ; OM, ou e memb ane.
No e also he mi ochond ion (Mi ) close o he cyanobac e ial cell.
ans e se sec ions o cyanobac e ial cells (Figu e 3A) o
longi udinal sec ions o cyanobac e ial ilamen s (Figu e 4A)
could be obse ed. The cyanobac e ial ilamen CB 1 in Figu e 3A
is somewha il ed so ha a ans e se sec ion shows a whole cell
and a agmen o i s adjacen cell. Twen y-eigh H. hauckii cells
we e imaged con aining one o h ee ilamen s o R. in acellula is
pe dia om cell (1.3 ±0.6 [mean ±SD]). In o al, 34
R. in acellula is ilamen s we e measu ed o cell dimensions o
he he e ocys and ege a i e cells. Conside ing he dimensions
summa ized in Table 1, he he e ocys s we e abou 2.5- o 3- old
la ge in olume han an a e age ege a i e cell. The numbe o
ege a i e cells pe ilamen was 1.9 ±1.2 (mean ±SD), which,
conside ing ha sec ioning o il ed ilamen s may hide some o
he cells in he TEM, is consis en wi h ea lie epo s o 3.5 cells
pe ilamen ound by luo escence mic oscopy (Capu o e al.,
2019;Tuo e al., 2021). In he ilamen s con aining h ee o ou
ege a i e cells, ape ing o he ilamen was e iden as he size
o he cells dec eased along he ilamen , wi h he smalles cell
being loca ed a hes om he e minal he e ocys (Figu e 4A
and Table 1). Because ilamen s could be somewha il ed in he
sec ioning plane, ape ing obse ed by TEM should be aken wi h
cau ion; howe e , his obse a ion is consis en wi h ape ing
o he R. in acellula is ilamen s isualized by luo escence
mic oscopy (Hil on e al., 2013;Pie ella Ka lusich e al., 2021).
Taking as e e ence a ilamen o R. in acellula is made o
one he e ocys and ou ege a i e cells (see cell dimensions
in Table 1) and a s anda d H. hauckii cell as iewed by TEM
(see Figu e 2: leng h ca. 25–30 µm, diame e ca. 15–20 µm;
conside ed as a cylinde o simpli y calcula ions), we es ima e ha
one cyanobac e ial ilamen occupies abou 1% o he olume o
he hos dia om.
The cyanobac e ial cell en elope, comp ising he cy oplasmic
memb ane, pep idoglycan laye and ou e memb ane, was e y
well de ined in many images (e.g., Figu e 3B). Addi ionally,
hylakoids (pho osyn he ic in acellula memb anes) we e
gene ally obse ed, and ca boxysomes (CO2- ixing s uc u es)
we e clea ly obse ed in some images (e.g., Figu e 4B).
Abundan glycogen g anules we e obse ed only in ew images
(Supplemen a y Figu e 4), indica ing ha he co esponding
H. hauckii-R. in acellulais cells we e in a di e en physiological
s a e han he o he cells ha we e isualized ha lacked such
a la ge numbe o glycogen g anules. In con as , no e idence
was ound o he p esence o gas esicles, which a e common in
o he oceanic ilamen ous cyanobac e ia (Hynes e al., 2012).
Independen ly o he sec ion, mi ochond ia we e equen ly
obse ed close o he cyanobac e ium (Figu es 3B,4B–D),
mo e equen ly associa ed wi h he ege a i e cells han
wi h he e ocys s and some imes accommoda ed nea by he
cyanobac e ial ilamen in he zone o he in e cellula sep a
(Figu e 4C). In some ins ances, he mi ochond ia we e igh ly
associa ed o he cyanobac e ial ou e memb ane (Figu e 4D
and see Supplemen a y Figu e 6 below). In spi e o an excellen
de ini ion o memb anes, no memb ane was obse ed in any
case su ounding a cyanobac e ial cell ou side o he ou e
memb ane o be ween he ou e memb ane and a nea by
mi ochond ion. These obse a ions s ongly suppo he idea
ha R. in acellula is esides di ec ly in he cy oplasm o
H. hauckii.
Richelia in acellula is En elope Vesicles
A dis inc ea u e obse ed in he ege a i e cells o
R. in acellula is was he p esence o esicles in hei pe iplasmic
space (Figu e 5 and see also Supplemen a y Figu e 5).
These s uc u es a e abou 40–60 nm in diame e , and hei
F on ie s in Mic obiology | www. on ie sin.o g 5Ma ch 2022 | Volume 13 | A icle 799362
micb-13-799362 Ma ch 11, 2022 Time: 16:49 # 6
Flo es e al. Dia om-Cyanobac e ium Ni ogen-Fixing Symbiosis
FIGURE 4 | T ansmission elec on mic og aph o mi ochond ia and R. in acellula is in he cy oplasm o H. hauckii.(A) Longi udinal sec ion o a R. in acellula is
ilamen (Rin ) composed o one e minal he e ocys (He ) and ou ege a i e cells (Vg ). The cyanobac e ial ilamen is loca ed wi hin a dia om cell; , us ule.
(B) T ans e se sec ion o a cyanobac e ial cell wi h mi ochond ia (Mi ) nex o he ou e memb ane (OM). No e also he p esence o ca boxysomes (Cbs) and
hylakoids (in acellula memb anes) in he cyanobac e ium. (C) Magni ied iew o a sec ion o he ilamen om panel (A) con aining ege a i e cells and
mi ochond ia (Mi ) nea by; OM, ou e memb ane. (D) T ans e se sec ion o a R. in acellula is cell showing nea by mi ochond ia (some indica ed by numbe s, 1–3).
No e ha mi ochond ia 1 and 2 a e igh ly associa ed o he ou e memb ane o he cyanobac e ium, whe eas mi ochond ion 3 is no bound o he cyanobac e ium.
esicula na u e is e iden as hey appea o be composed o
a closed bilaye . Thei localiza ion in he pe iplasm sugges s
ha hese esicles o igina e om he cy oplasmic memb ane.
These esicles a e mainly ound ou side o he pep idoglycan
laye and wi hin pa ial e agina ions o he ou e memb ane.
Ou side o he ou e memb ane, some double esicles abou
100 nm ×150 nm (eVs) o 115 nm in diame e (eVs∗) ha
consis o one o wo esicles su ounded by a memb ane we e
obse ed (Figu e 5). These double esicles may ep esen esicles
ha ha e been ex uded om he cyanobac e ium su ounded
by ou e memb ane. Finally, some small esicles obse ed
in he dia om’s cy oplasm close o he cyanobac e ium ( Vs)
migh co espond o eleased pe iplasmic esicles (Figu e 5 and
Supplemen a y Figu e 5). I his we e he case, eleased esicles
wi h one memb ane ( Vs) could ep esen ex uded esicles ha
shed he ou e memb ane laye .
Dis inc He e ocys Mo phology
In he Richelia ilamen s shown in Figu es 2A,4A, he
he e ocys s p esen a cell en elope ha is hinne han ha
usually obse ed in ee-li ing he e ocys - o ming cyanobac e ia
such as Anabaena, abou 100 nm wide in R. in acellula is
s. 250–600 nm wide in Anabaena (see, e.g., Flo es e al.,
2019). In con as , he en elope o he ege a i e cells is
abou 50 nm wide in bo h R. in acellula is (Figu es 3B,5)
and Anabaena (Wilk e al., 2011). The blank space a ound
a he e ocys , as seen in Figu e 4A, is a equen a i ac
ha , in he he e ocys s o Anabaena, esul s om sepa a ion
o he Hgl laye om he ou e memb ane du ing sample
p epa a ion (see, e.g., Flo es and He e o, 2010;Flo es e al.,
2019). O he examples o he e ocys s ha show a ela i ely
hin, bu well labeled en elope, a e shown in Figu es 6A,C. On
he o he hand, in con as o Anabaena he e ocys s in which
in acellula memb anes end o concen a e a he cell poles
o ming he honeycomb s uc u e nex o he he e ocys neck
(see, e.g., Flo es and He e o, 2010;Flo es e al., 2019), he
Richelia he e ocys s equen ly showed in acellula memb anes
h oughou (Figu es 4A,6C). None heless, he he e ocys in
Figu e 6A pa ially shows a honeycomb s uc u e as well as
a cyanophycin g anule, ypically ound nea by he he e ocys
F on ie s in Mic obiology | www. on ie sin.o g 6Ma ch 2022 | Volume 13 | A icle 799362
micb-13-799362 Ma ch 11, 2022 Time: 16:49 # 7
Flo es e al. Dia om-Cyanobac e ium Ni ogen-Fixing Symbiosis
FIGURE 5 | T ansmission elec on mic og aphs o sec ions o a ege a i e cell o R. in acellula is endosymbio ic in H. hauckii showing nume ous cell
en elope- ela ed esicles. Pe iplasmic esicles mainly loca ed in ou e memb ane e agina ions (Vs) and possible ecen ly eleased esicles ( Vs) can be obse ed.
Two closely loca ed esicles (2Vs) could be ex e nalized oge he gi ing ise o an ex uded esicle such as ha shown wi h wo in e nal esicles (eVs∗). Two
mi ochond ia nea by he cyanobac e ial ou e memb ane (OM) a e also indica ed (Mi ). In he lowe - igh mic og aph, a possible ex uded esicle con aining one
in e nal esicle is obse ed (eVs).
neck. Likely because R. in acellula is occupies a small olume,
a clea honeycomb s uc u e and he e ocys neck, including
he cyanophycin g anule, we e obse ed only in one specimen
(Figu e 7). This obse a ion indica es he p esence o hese
s uc u es in he he e ocys s o R. in acellula is.
In R. in acellula is, he ou e memb ane is
con inuous be ween ege a i e cells (Figu e 4C and
Supplemen a y Figu es 6A,B) and be ween he he e ocys
and i s adjacen ege a i e cell (Figu es 6A,7and
Supplemen a y Figu e 7A), as is usual in he e ocys - o ming
cyanobac e ia (Flo es e al., 2006, 2016;Wilk e al., 2011). As
obse ed in di e en samples, he R. in acellula is he e ocys
en elope con ains i e laye s including he cy oplasmic and ou e
memb anes. Impo an ly, he con inui y o he ou e memb ane
wi h ha o he adjacen ege a i e cell shows ha he ou e
memb ane is he ou e -mos laye o he he e ocys en elope
in R. in acellula is. In addi ion o he pep idoglycan laye , wo
addi ional laye s o unknown composi ion can be disce ned nex
o he cy oplasmic memb ane (mo e clea ly seen in Figu e 6D
han in Figu e 6B and see also Supplemen a y Figu es 7B,D).
Because he s aining p ocedu e used was based on educed
osmium e oxide (see “Ma e ials and Me hods”), which labels
lipids e icien ly (Tapia e al., 2012), we hypo hesize ha he
sha ply labeled laye con ains lipids and, he e o e, could
cons i u e he he e ocys glycolipid laye ( en a i ely ma ked
as Hgl in Figu e 6 and Supplemen a y Figu e 7). The laye
nex o, inside he ou e memb ane, could cons i u e he
he e ocys polysaccha ide laye ( en a i ely ma ked as Hep in
Figu e 6 and Supplemen a y Figu e 7). Thus, in con as o
he en elope o he he e ocys s o ee-li ing he e ocys - o ming
cyanobac e ia, he he e ocys s o R. in acellula is lack an
ex a en elope ou side o he ou e memb ane. Ins ead, he
componen s o he he e ocys -speci ic en elope seem o be
loca ed in he pe iplasmic space, al hough his hypo hesis will
need co obo a ion.
Possible Thi d Pa ne in he Hemiaulus
hauckii Symbiosis
In one H. hauckii cell, a second possible p oka yo ic o ganism
was obse ed loca ed in he cy oplasm (Figu e 8A). I consis ed
o a g oup o cells wi h di e en sizes, and he numbe o cells
ha could be seen depended on he sec ioning plane. Thus,
wo cells a e seen in panel A, h ee in panel B, and i e in
panel C (Figu e 8; see ull se ies o images o his specimen in
Supplemen a y Figu e 8). The whole g oup o cells is ema kably
small, abou 1.8-µm wide. Assuming a sphe ical o m, his g oup
o cells is es ima ed o ep esen only abou 0.03% o he hos cell
olume. The ul as uc u e o his o ganism (Figu e 8) esembles
F on ie s in Mic obiology | www. on ie sin.o g 7Ma ch 2022 | Volume 13 | A icle 799362
micb-13-799362 Ma ch 11, 2022 Time: 16:49 # 8
Flo es e al. Dia om-Cyanobac e ium Ni ogen-Fixing Symbiosis
FIGURE 6 | T ansmission elec on mic og aphs o R. in acellula is he e ocys s and de ails o he he e ocys en elope. (A) Magni ied iew o he le -hand he e ocys
(He ) in Figu e 2A. No e he ou e memb ane (OM), which is con inuous wi h ha o he adjacen ege a i e cell, and he p esence o a possible cyanophycin g anule
(CG); he ela i e accumula ion o memb anes close o he CG esembles he he e ocys honeycomb s uc u e. (B) Magni ied iew o pa o he en elope om he
he e ocys shown in panel (A) (do ed squa e). No e he p esence o cy oplasmic memb ane (CM) and ou e memb ane (OM). Th ee addi ional laye s en a i ely
iden i ied as pep idoglycan laye (PG), glycolipid laye (Hgl) and polysaccha ide laye (Hep) a e shown. (C) Pa o a dia om cell in which a he e ocys (He ) and pa o
an adjacen ege a i e cell (Vg ) can be obse ed. No e he p esence o nea by mi ochond ia (Mi ). (D) Magni ied iew o pa o he en elope om he he e ocys
shown in panel (C) (do ed squa e). He e ocys en elope laye s en a i ely iden i ied as cy oplasmic memb ane (CM), pep idoglycan laye (PG), glycolipid laye (Hgl),
polysaccha ide laye (Hep) and ou e memb ane (OM) a e indica ed. No e ha he pu a i e glycolipid laye is hinne in his image han in ha shown panels (A,B).
ha o cyanobac e ia o he genus Ch oococcidiopsis, which
ep oduce by bo h bina y and mul iple ission p oducing cells o
di e en sizes (Wa e bu y and S anie , 1978;Billi e al., 2001).
Th ee en elope laye s could be disce ned (Figu e 8B), sugges ing
a G am-nega i e ype o cell en elope. This is consis en wi h
he possibili y ha his is a cyanobac e ium in which an ou e
memb ane (laye L3; Figu e 8B) is sha ed by all he cells
in he g oup. Unlike cyanobac e ia, howe e , his o ganism
con ains, ins ead o ypical hylakoids, cy oplasmic esicles ha
a e abou 50 nm in diame e and esemble he ch oma opho es o
pho osyn he ic bac e ia such as Rhodobac e sphae oides (Noble
e al., 2018). As in he case on R. in acellula is, a mi ochond ion
is obse ed igh ly associa ed o he ou e memb ane o his
second cy oplasmic o ganism (Figu e 8C). Addi ionally, some
esicles a e obse ed in he pe iplasmic space and ou side o he
cell g oup (Figu e 8B), esembling he esicles desc ibed ea lie
o R. in acellula is.
DISCUSSION
In his wo k, we ha e shown ha in he Hemiaulus hauckii-
Richelia in acellula is symbiosis, he endosymbion e ains
he G am-nega i e ype o cell en elope cha ac e is ic o
cyanobac e ia and esides di ec ly in he cy oplasm o he dia om
(Figu e 9). Hemiaulus hauckii can also hos a second p oka yo ic
o ganism in i s cy oplasm ha shows some esemblance o
cyanobac e ia o he genus Ch oococcidiopsis. Because o i s small
es ima ed size and he na u e o TEM, hese o he cells could
ha e been la gely unde ec ed in ou samples, which un o una ely
makes es ima ing he equency o i s p esence di icul . Thus, we
do no know whe he hese obse a ions ep esen an occasional
associa e o H. hauckii (e.g., a pa hogen) o an es ablished
hi d pa ne in he symbiosis. In e es ingly, isualiza ion
by epi luo escence mic oscopy has shown he p esence o a
possible second non- ilamen ous cyanobac e ial endosymbion
F on ie s in Mic obiology | www. on ie sin.o g 8Ma ch 2022 | Volume 13 | A icle 799362
micb-13-799362 Ma ch 11, 2022 Time: 16:49 # 9
Flo es e al. Dia om-Cyanobac e ium Ni ogen-Fixing Symbiosis
in H. hauckii collec ed om he same loca ion as he cells
p esen ed he e (see small luo escence spo apa om he
luo escen Richelia ilamen s in Figu e 1B o Hil on e al., 2013)
as well as om a di e en loca ion (Supplemen a y Figu e 9).
To he bes o ou knowledge, hese obse a ions ep esen he
i s epo o a possible addi ional pa ne in hese globally
dis ibu ed symbioses, and hus wa an u he in es iga ion.
The loca ion o R. in acellula is in he cy oplasm o H. hauckii
should in luence he mechanism(s) o molecula exchange
be ween he wo pa ne s. The cyanobac e ium ixes CO2
and N2(Fos e e al., 2021), and i has o ob ain nu ien s
necessa y o i s g ow h om he dia om’s cy oplasm. A ecen
e iew o memb ane anspo e s encoded in he genome o
R. in acellula is HH01, an endosymbion o a H. hauckii ha
was isola ed om he same loca ion as he H. hauckii s udied
he e (Hil on e al., 2013), has iden i ied a numbe o pu a i e
anspo e s o suga s, amino acids, sul u , phospho us, me als
and i amins ha can suppo he g ow h o he cyanobac e ium
(Nie es-Mo ión e al., 2020). Richelia in acellula is p o ides
H. hauckii wi h ixed ni ogen (Fos e e al., 2011), and he e o e
mechanism(s) o molecula ans e (in pa icula , o ixed
ni ogen) om he cyanobac e ium o he dia om mus also exis .
Howe e , possible memb ane expo e s encoded in he genome
o R. in acellula is HH01 we e no as e iden o nume ous as
memb ane impo e s (Nie es-Mo ión e al., 2020), sugges ing
he need o ano he s a egy o me aboli e expo . He e, we
ha e shown ha R. in acellula is p oduces cell en elope esicles,
eadily obse ed in he pe iplasm o ege a i e cells be o e
being p esumably ex e nalized su ounded by a agmen o
he ou e memb ane. These esicles esemble he ou e -inne
memb ane esicles o G am-nega i e bac e ia which can ca y
cy oplasmic ma e ial (Gill e al., 2019;Toyo uku e al., 2019).
We hypo hesize ha he memb ane esicles su ounding Richelia
can con ibu e o he ans e o nu ien s (especially ixed
ni ogen) om he cyanobac e ium o i s hos . In e es ingly,
memb ane esicles ha e been obse ed in he p oximi y o
he pe iplasmic symbion o he dia om Rhizosolenia cle ei
(Janson e al., 1995) and a cyanobac e ial symbion in a lichen
(Pe eling, 1973). Memb ane esicles ha e also been sugges ed o
be in ol ed in expo o cy oplasmic ma e ial om Anabaena
cells o he g ow h medium (Oli ei a e al., 2015). Ou e -inne
memb ane esicles can also ca y mac omolecules including
nucleic acids (Gill e al., 2019), and memb ane esicles con aining
DNA ha e been obse ed in he cyanobac e ial symbion o
he e n Azolla (Zheng e al., 2009). The size o he genome
o R. in acellula is HH01 is abou 3.24 Mbp, much smalle
han he genome size o mos ee-li ing he e ocys - o ming
cyanobac e ia, which is abou 7–9 Mbp (Hil on e al., 2013). I
some cyanobac e ial genes ha e been ans e ed o he dia om
nucleus, as has happened in he de elopmen o endosymbion -
de i ed o ganelles (Bock, 2017), he memb ane esicles could
ep esen a ehicle o gene ans e .
N2 ixa ion is e y sensi i e o oxygen, and in R. in acellula is
he N2 ixa ion machine y is likely exposed no only o ambien
oxygen bu also o oxygen p oduced pho osyn he ically by bo h
i s ege a i e cells and hos dia om. He e we ha e obse ed he
p esence o nume ous mi ochond ia nea by he cyanobac e ia
FIGURE 7 | T ansmission elec on mic og aph o he ege a i e
cell-he e ocys junc ion in a R. in acellula is ilamen endosymbio ic in
H. hauckii. No e he honeycomb memb ane s uc u e (HC), he he e ocys
neck, he con inuous ou e memb ane (OM) and he cyanophycin g anule
(CG).
in he dia om’s cy oplasm. Al hough his migh e lec he
p esence o a high numbe o mi ochond ia in he dia om,
mi ochond ia equen ly appea ed o be igh ly associa ed o he
ou e memb ane o he cyanobac e ium, e ealing a possible
speci ic ela ion. I is possible ha he mi ochond ia con ibu e o
dec easing he le el o in acellula oxygen in he icini y o he
cyanobac e ium. Whe he he endosymbion could addi ionally
in e ac wi h he hos mi ochond ia as has been shown o some
endosymbio ic algae (Song e al., 2017;Uwizeye e al., 2021) and
o he ick bac e ial symbion Midichlo ia mi ochond ii (S a u
e al., 2020), o al e he me abolism o he mi ochond ia as has
been desc ibed o pa hogenic bac e ia ha a ge he o ganelles
o euka yo ic cells (Escoll e al., 2016), is unknown.
F on ie s in Mic obiology | www. on ie sin.o g 9Ma ch 2022 | Volume 13 | A icle 799362
