Alkaline phosphatases in microbialites and bacterioplankton from Alchichica soda lake, Mexico

R E S EA RCH AR T I C L E

Alkaline phosphatases in microbialites and bacterioplanktonfrom Alchichica soda lake, Mexico

Patricia M. Valdespino-Castillo1,2, Rocio J. Alc�antara-Hern�andez1,2, Javier Alcocer3,Mart�ın Merino-Ibarra4, Miroslav Macek3,5 & Luisa I. Falc�on2

1Posgrado en Ciencias del Mar y Limnolog�ıa, Universidad Nacional Aut�onoma de M�exico, Coyoac�an, Mexico; 2Laboratorio de Ecolog�ıa Bacteriana,

Instituto de Ecolog�ıa, Universidad Nacional Aut�onoma de M�exico, Coyoac�an, Mexico; 3Proyecto de Investigaci�on en Limnolog�ıa Tropical, FES

Iztacala, UNAM, Tlalnepantla, Estado de M�exico, M�exico; 4Unidad Acad�emica de Ecolog�ıa y Biodiversidad Acu�atica, Instituto de Ciencias del Mar

y Limnolog�ıa, Universidad Nacional Aut�onoma de M�exico, Coyoac�an, M�exico; and 5Academy of Sciences of the Czech Republic, Biology Centre

v. v. i. Institute of Hydrobiology, �Cesk�e Bud�ejovice, Czech Republic

Correspondence: Luisa I. Falc�on, Instituto de

Ecolog�ıa, Universidad Nacional Aut�onoma de

M�exico, Circuito exterior sn, Cd.

Universitaria, Coyoac�an 04510, Mexico.

Tel.: +52 55 5622 8222, ext. 46869;

fax: +52 55 5622 8995;

e-mail: [email protected]

Received 1 February 2014; revised 6 August

2014; accepted 7 August 2014.

DOI: 10.1111/1574-6941.12411

Editor: Gary King

Keywords

dissolved organic phosphorus utilization;

extracellular enzymes; low calcium

environment; microbial functional diversity.

Abstract

Dissolved organic phosphorus utilization by different members of natural com-

munities has been closely linked to microbial alkaline phosphatases whose affil-

iation and diversity is largely unknown. Here we assessed genetic diversity of

bacterial alkaline phosphatases phoX and phoD, using highly diverse microbial

consortia (microbialites and bacterioplankton) as study models. These micro-

bial consortia are found in an oligo-mesotrophic soda lake with a particular

geochemistry, exhibiting a low calcium concentration and a high Mg : Ca ratio

relative to seawater. In spite of the relative low calcium concentration in the

studied system, our results highlight the diversity of calcium-based metallo-

phosphatases phoX and phoD-like in heterotrophic bacteria of microbialites

and bacterioplankton, where phoX was the most abundant alkaline phosphatase

found. phoX and phoD-like phylotypes were more numerous in microbialites

than in bacterioplankton. A larger potential community for DOP utilization in

microbialites was consistent with the TN : TP ratio, suggesting P limitation

within these assemblages. A cross-system comparison indicated that diversity of

phoX in Lake Alchichica was similar to that of other aquatic systems with a

naturally contrasting ionic composition and trophic state, although no phylo-

types were shared among systems.

Introduction

Phosphorus is an essential element for cellular structure

and function. Its low availability in natural systems

(Wu et al., 2000) and its high turnover rates (Benitez-

Nelson & Buesseler, 1999; Benitez-Nelson, 2000; Ammer-

man et al., 2003) explain its demand by living systems and

correspond with a diverse metabolic network that has been

widely distributed in different life forms. We are starting

to recognize the identity of the organisms that harbour the

enzymatic tools for P transformations, among which

microbes, particularly heterotrophic bacteria (Dyhrman

et al., 2007; Cunha et al., 2010), play a central role (Kono-

nova & Nesmeyanova, 2002; White, 2009).

In oligotrophic waters, dissolved organic phosphorus

(DOP) typically constitutes the major fraction of total

dissolved P. Thus, regeneration of P from DOP has been

shown to play a significant role in supplying the P

required for biological production (Karl & Bj€orkman,

2001, 2002; Dyhrman et al., 2006; Young & Ingall, 2010),

particularly when the availability of dissolved inorganic

phosphorus (DIP) is low (e.g. in the upper layer of strati-

fied water bodies). Phosphomonoesters are an important

fraction of DOP in aquatic environments (Young &

Ingall, 2010). The degradation of these compounds

requires the action of phosphatases (Harke et al., 2012),

pH-dependent metalloenzymes (Jansson et al., 1988). The

catalytic activity of these enzymes is part of the initial

steps of organic matter degradation (Pinchuk et al.,

2008), where orthophosphate is cleaved hydrolytically

from organic molecules (Jansson et al., 1988). The

activity of these enzymes in aquatic systems has been

FEMS Microbiol Ecol && (2014) 1–16 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

MIC

ROBI

OLO

GY

EC

OLO

GY

associated to bacterioplankton, phytoplankton (Jansson

et al., 1988; �Strojsov�a et al., 2003; Yamaguchi & Adachi,

2010) and zooplankton (Vidal et al., 2003), although their

diversity and distribution remain largely unknown

(Tringe et al., 2005; Cunha et al., 2010).

Recent studies, based on the amplification of genetic

regions associated to P-enzymes, have shown a differential

abundance and distribution of alkaline phosphatases (AP)

among microbial assemblages (Sakurai et al., 2008; Sebas-

tian & Ammerman, 2009). Alkaline phosphatase PhoA

has been widely described in cultured organisms such as

Escherichia coli. However, in an analysis of the Global

Ocean Survey dataset, Sebastian & Ammerman (2009)

proposed PhoX as a novel alkaline phosphatase more

broadly distributed in marine bacteria than the ‘classical

PhoA’. Soon after, Luo et al. (2009) reported that PhoD

was the most frequently AP found in marine environ-

ments. Along with these results, the availability of differ-

ent metal cofactors has been linked to the enzymatic

activity, and the potential presence and distribution of

AP (Monds et al., 2006; Wu et al., 2007; Luo et al., 2009;

Kathuria & Martiny, 2011). Ca+2 is a cofactor of

AP-PhoX and PhoD, whereas Mg+2 and Zn+2 are cofac-

tors of PhoA. In this sense, the low presence of phoA

genes could be related to low availability of Zn+2 in the

open ocean (sensu Luo et al., 2009). Although the infor-

mation about the presence and abundance of these genes

is growing, exploration of the diversity and dynamics of

AP in natural systems is still needed.

To gain insight into AP diversity, we used complex

microbial consortia (microbialites and bacterioplankton)

as study models from an oligo-mesotrophic (sensu Alco-

cer et al., 2000; Macek et al., 2009) environment with a

low phosphorus concentrations. The cationic proportions

of the saline alkaline Lake Alchichica (Fig. 1) differ signif-

icantly from those of seawater, particularly of Ca+2, Mg+2,

and Zn+2, displaying a peculiar chemistry characterized

by low calcium concentrations and a high Mg : Ca ratio

N

sM S3

400 m

sM S2

sM S4

sM S1

sM S5

sM S6

cM S3

cM S6

cM S5 cM S1,S4

cM S2 W (5, 25, 61 m)

Fig. 1. Microbialite and bacterioplankton sampling sites in Lake Alchichica, Puebla, highlands of Central Mexico. Outlined squares represent

spongy-type microbialites (subsample sites 1–6); white squares, columnar-type microbialites (subsamples 1–6) and the green circle shows the

water column bacterioplankton sampling sites (5, 25 and 61 m).

FEMS Microbiol Ecol && (2014) 1–16ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

2 P.M. Valdespino-Castillo et al.

(Armienta et al., 2008; Ka�zmierczak et al., 2011; Mancil-

la-Villa et al., 2012), which resembles that of the ancient

soda ocean (sensu Kempe & Degens, 1985; Ka�zmierczak

et al., 2011).

Saline alkaline systems are habitats of a variety of

microorganisms, which have shown ecological and bio-

technological relevance (Antony et al., 2013). We

explored the distribution and diversity of functional genes

(alkaline metallophosphatases for DOP utilization) within

microbial communities (bacterioplankton and microbia-

lites) of Lake Alchichica to contribute to the understand-

ing of the interactions between the biogeochemical

phosphorus cycle (and therefore carbon cycle) and the

dynamics of other elements, as well as the identification

of AP-harbouring bacteria in this particular environment.

Materials and methods

Study area

Alchichica is a crater-lake located in the Central Mexican

Plateau (19°240N, 97°240W; 2340 m a.s.l.; Fig. 1) that

belongs to a cluster of six maar lakes on the easternmost

portion of the Trans-Volcanic Belt. The lake is saline

(8.5 g L�1), alkaline (pH 9.5) and deep (Zmean = 40.9 m

and Zmax = 62 m). The thermal regime of Lake Alchichi-

ca is warm-monomictic; the circulation period typically

occurs during the dry winter and the lake stratifies from

April to December. The ionic abundances of Lake Alchi-

chica are: Na+ > Cl� > Mg+2 > SO�24 > K+ > Ca+2 and

differ significantly from those of seawater, particularly

those of Ca+2, Mg+2, and Zn+2, because calcium concen-

tration is quite low (c. 0.3 mM) and there is a high

Mg : Ca ratio (c. 50; Armienta et al., 2008; Ka�zmierczak

et al., 2011). The study of this lake is now part of a

Long-Term Ecological Research program conducted by

UNAM (National Autonomous University of Mexico),

and its geological, hydrological and biological characteris-

tics have been described (Vilaclara et al., 1993; Lugo

et al., 1998; Alcocer et al., 2000).

The microbial assemblages of Lake Alchichica have also

been studied. Macek et al. (2009) surveyed the water-

column picoplankton and showed that total and auto-

trophic picoplankton dynamics follow a regular pattern

linked to the hydrodynamic cycle of the system. Larger

picoplankton peaks occur during circulation and early

stratification (January–March) and low numbers occur

during late stratification. Hern�andez-Avil�es et al. (2010)

identified, by FISH, a vertical zonation of bacterioplank-

ton components associated to diverse biogeochemical

processes, among which denitrification emerged as a

major pathway for N loss from the water body. Bautista-

Reyes & Macek (2012) applied CARD-FISH to describe

bacterial taxon annual shifts in abundance patterns and

their selective feeding by ciliates and heterotrophic flagel-

lates throughout the water column.

A discontinuous ring of microbialites is located along

the perimeter of this 2.26-km2 lake, where two different

morphologies have been reported and described as

spongy-type (white) and columnar-type (brown) sensu

Tavera & Kom�arek (1996) and Ka�zmierczak et al. (2011;

Fig. 2). The mineralogy, texture, isotopic features and

microbial composition are described in Ka�zmierczak et al.

(2011) and Centeno et al. (2012). The high Mg : Ca ratio

Fig. 2. Morphology of microbialites studied in Lake Alchichica: spongy microbialite (upper) and columnar microbialite (lower). The external

morphology of the exposed (emerged) microbialites is shown on left; a section of submerged microbialites (at a depth c. 0.3 m) is shown on the

right.


Phosphorus utilization by complex communities 3

(48.1 : 69.3) of Alchichica water is reflected in the micro-

bialite mineralogy, where uncommon hydromagnesite-

magnesite minerals are present (Ka�zmierczak et al., 2011).

Besides its structural complexity (Fig. 2), Alchichica

microbialites have shown important activity related to the

N cycle in a system that has been proposed to be limited

mainly by this element (Ram�ırez-Olvera et al., 2009;

Ardiles et al., 2012). Microbialites have shown high N2

fixation rates produced primarily by heterocystous

cyanobacteria (Falc�on et al., 2002; Beltr�an et al., 2012).

Sampling

Samples were taken during well established circulation

(February) and stratification (August) periods in 2011

from the two morphological types of microbialites that

coexist in the perimeter of Lake Alchichica. Microbialite

samples of c. 10 g were taken at a depth of c. 0.30 m

from six sampling sites distributed throughout the perim-

eter of the lake (Fig. 1). Six samples were collected for

each microbialite morphological type (columnar and

spongy) per triplicate and per site during well established

lake circulation and during stratification. Spongy micro-

bialites are more widely distributed than columnar micro-

bialites, which are concentrated around the W–SW third

of the lake shoreline (Fig. 1). Samples were frozen at

�20 °C until DNA extraction.

Bacterioplankton samples were taken at the centre of

the lake (Fig. 1) from three water depths: 5, 25 and

61 m, corresponding to the epilimnion, metalimnetic base

(during stratification) and bottom water (1 m above the

sediment), respectively. The same depths were sampled

during well established lake circulation. Triplicate 0.5-L

water samples were filtered through Osmonics (Poretics

Corp.) polycarbonate membranes (pore size 0.22 lm) per

triplicate for each depth. Filters were placed in DNA-free

2.0 mL tubes and kept frozen at �20 °C until analysis.

Environmental characterization

Physicochemical characterization of water column sam-

ples was performed to frame alkaline phosphatase poten-

tial. Temperature, dissolved oxygen (DO), pH and

conductivity of the water column and of the water sur-

rounding the microbialites, were recorded with a YSI

6600 multiparameter probe. Water samples for nutrient

analysis including soluble reactive phosphorus (SRP),

NO�2 , NO�

3 , dissolved inorganic nitrogen (DIN), total

phosphorus (TP), and total nitrogen (TN) were collected

in the same sites and kept in polypropylene containers

after filtration through 0.45 and 0.22 lm (HA Milli-

poreTM) mixed cellulose esters membranes. Samples were

kept frozen until their analysis (within 24–48 h) with a

segmented flow Autoanalyzer (Skalar San-plus) using the

standard methods adapted by Grasshoff et al. (1983) and

the circuits suggested by Kirkwood (1994). Organic P was

calculated as TP minus SRP.

A subsample of 1 cm2 of area (from the surface to

approximately first 2 cm depth) of each microbialite was

excised, lyophilized (Savant SpeedVac drier, Waltham,

MA), and then ground in an agate mortar and pestle for

further analysis. A homogeneous subsample of 0.1 g of

each microbialite sample was analysed to quantify total

phosphorus and total nitrogen after high-temperature

persulphate oxidation (Valderrama, 1981) and to assess

the nutrient limitation condition of the microbialite.

Although microbialite samples were heterogeneous, in

each case they comprised 1 cm2 of surface-living tissue

and a fraction of mineral matrix.

DNA extraction

DNA was extracted from the six different sites for each

type of microbialite (spongy and columnar) and pooled

to account for the global composition of each microbia-

lite type. Microbialite DNA extraction was carried out

using the protocol of Zhou et al. (1996), modified for

microbialite samples (Centeno et al., 2012). Approxi-

mately 5 g of each microbialite was pulverized along with

extraction buffer (100 mM Tris-HCl, 20 mM NaCl,

100 mM EDTA, pH 8) and 0.06 V of cetyl trimethylam-

monium bromide (CTAB) in liquid nitrogen. The

obtained mixture was incubated with lysozyme

(30 mg mL�1; Sigma Aldrich) for 30 min at 37 °C and

subsequently with proteinase K (10 mg mL�1; Sigma

Aldrich) and 0.1 V of sodium dodecyl sulphate (SDS; at

55 °C, overnight). Samples were then centrifuged at

1800 g, 20 min. The aqueous phase was recovered and

extracted twice with phenol : chloroform : isoamyl alco-

hol (25 : 24 : 1) and once with chloroform : isoamyl

alcohol (24 : 1). DNA was precipitated overnight at

�20 °C by adding 2 V of 2-propanol, 0.1 V of 3 M

sodium acetate (3M) and 2 lL of GlycoBlue (Ambion

Inc.). The precipitated DNA was recovered by centrifuga-

tion (8000 g, 15 min), washed twice with 80% ethanol

and resuspended in molecular grade water. The DNA

samples were then purified through Mini Spin columns

of DNeasy Blood & Tissue kit (QIAGEN, Alameda, CA)

according to the manufacturer’s instructions, and precipi-

tated again as mentioned above. Purified DNA was stored

at �20 °C until analysis.

Alkaline phosphatase amplification

A region of c. 400 bp of the phoD gene was PCR-ampli-

fied following the protocol of Sakurai et al. (2008) with



the primers ALPS-F730 (50-CAGTGGGACGACCACGAGGT-30) and ALPS-R1101 (50-GAGGCCGATCGGCATGTCG-30). PCR was performed using c. 10 ng of

DNA in a final reaction mixture of 25 lL, containing

2 lM of each primer, 2.5 lL 109 ViBuffer S (Vivantis,

Oceanside, CA), 0.2 mM of each dNTP and 0.5 U of Taq

DNA Polymerase (Vivantis). Amplification steps com-

prised an initial denaturation step at 94 °C for 3 min, 35

cycles of a denaturing step at 94 °C for 1 min, annealing

at 59 °C for 1 min, and extension at 72 °C for 2 min,

followed by a final extension at 72 °C for 7 min and

cooling at 4 °C.A fragment of c. 600 bp of the phoX gene was amplified

from environmental DNA using F/R primers phoX1, phoX2

and phoX3 (Sebastian & Ammerman, 2009): phoX1-F

(50-GARGARAAYTTYAACGGCTA-30) and phoX1-R (50-GCCAKSACRWAVAGATCC-30); phoX2-F (50-GARGAGAACWTCCACGGYTA-30) and phoX2-R (50-GATCTCGATGATRTGRCCRAAG-30); phoX3-F (50-GGGNACTTAYYT-MACBTGYGAA-30) and phoX3-R (50-GDCKATCCATBGKBGTTGC-30). PCR was performed using c. 10 ng of

DNA in a final reaction mixture of 25 lL, containing

0.4 lM of each primer, 2.5 lL 109 ViBuffer S (Vivantis),

0.2 mM of each dNTP and 0.5 U of Taq DNA Polymerase

(Vivantis). The PCR program comprised an initial dena-

turation at 94 °C for 5 min, 35 cycles of 94 °C for 30 s,

annealing at 52 °C for 30 s, extension at 72 °C for 1 min,

final extension of 72 °C for 10 min and cooling at 4 °C.PCR products from five reactions were pooled and then

gel-purified using QIAquick spin columns (Qiagen). Clone

libraries were constructed for each sample (per microbia-

lite morphology and water column sampling site) and sea-

son. Amplified phoD and phoX fragments were ligated to

pCR� 2.1 vector using the Original TA Cloning Kit (Invi-

trogen, Carlsbad, CA). Chemically competent E. coli TOP-

10 cells were transformed and selected using LB plates with

ampicillin (100 lg mL�1) and X-Gal (80 lg mL�1);

clones were then further screened by PCR, using M13

primers.

Sequence analysis

Sequence alignment for functional genes was done with

the predicted amino acid sequences aligned with CLUSTAL

W to which DNA sequences were imposed to avoid inser-

tions, deletions, internal stop codons or reading frame

shifts. A BLAST similarity search of problematic sequences

was used to exclude pseudogenes from the analysis. The

nucleotide sequences obtained from microbialites and

bacterioplankton were BLAST-searched using the BLASTN

tool (\http://www.ncbi.nlm.nih.gov/Blast.cgi) in the

National Center for Biotechnology Information (NCBI)

database. Sequences of the entire dataset were aligned

with SEQUENCHER 4.1.4 (Gene Codes Corp., Ann Arbor,

MI). Maximum likelihood branch lengths were fitted to

consensus trees with PHYML 3.0 (Guindon et al., 2010).

Maximum likelihood bootstrap values were inferred from

1000 replicates. Phylogenetic topologies were visualized

and edited with FIGTREE (v.1.3.1). The determined AP par-

tial gene sequences were deposited in the GenBank data-

base under accession numbers KF891484–KF891515,KF891517–KF891828, KF891830–KF891882.

Operational taxonomic unit (OTU)-based analyses

from MOTHUR v.1.33.3 platform (Schloss et al., 2009)

were used to estimate diversity and coverage for the

selected genetic markers. Distance matrices and OTUs

for each set of sequences were calculated with the func-

tions ‘dist.seqs’ and ‘cluster’ using the furthest neighbour

algorithm. The cutoff value for phoX analysis was 0.04,

considered a reference criteria for defining OTUs of

functional genes (sensu Iwai et al., 2011). The phoD cut-

off value was 0.25, as estimated recently by Tan et al.

(2013). A nonparametric richness estimator (Chao) and

the Shannon diversity index (H0) were obtained for the

sets of sequences of Lake Alchichica but also for the sets

of sequences (phoX) found in different environmental

studies (see Table 1).

Results

A total of 398 AP novel sequences were recovered from

the environmental DNA extracted from microbialites and

bacterioplankton of Alchichica soda-lake. Water environ-

ment showed relatively high P concentrations and seemed

to be N limited, at least during the sampling dates

reported here (Table 2). In contrast, the TN : TP ratio of

microbialites, which integrates over a longer period,

points to P limitation in these consortia.

In this relatively low calcium system (high Mg : Ca

ratio), the abundance of calcium-based AP phoX was

higher overall than the phoD-like set of sequences, as can

be observed by the number of OTUs found (and also by

the number of clones; Table 1).

Physicochemical characterization and nutrient

status

Physicochemical parameters of the water surrounding mi-

crobialites (average of 12 sampled sites) are summarized

(Table 2), for circulation (February) and stratification

(August) periods along with environmental conditions in

the three water-column depths sampled for bacterioplank-

ton analysis.

Nutrients, TP and TN varied between stratification and

circulation (Table 2). Among the physicochemical parame-

ters measured, DO and pH showed a higher heterogeneity



Table

1.Bacterial

APphoD

andphoX

ofmicrobialassemblages

analysed

inthis

study:

microbialites

andbacterioplanktonofLake

Alchichica(duringcirculationan

dstratificationperiods)

and

environmen

taldatasetsfrom

differentphoXsurveys(Atlan

ticseaw

ater,mixed

water

andamicrocosm

experim

entin

Lake

Taihu,China)

System

Bacterial

assemblage

Season

phoD

phoX

No.sequen

ces

obtained

Unique

sequen

ces

OTU

s*Chao

Shan

non

(H0 )

Coverage

(%)

No.sequen

ces

obtained

Unique

sequen

ces

OTU

s†Chao

Shan

non

(H0 )

Coverage

(%)

Lake Alchichica‡

Microbialites

Circulation

39

15

20.0

2.22

92

50

16

30.0

2.28

88

Stratification

30

13

20.5

1.68

92

31

77.0

1.67

100

Bacterioplankton

Circulation

65

8.0

1.56

33

35

77.3

1.15

97

Stratification

19

77.3

1.44

93

11

616.0

1.04

72

Total

180

94

23§

68

2.16

94

192

120

27§

53.0

2.36

93

Sargasso

Seaan

d

Chesap

eake

Bay

¶Seaw

ater

––

––

–57

54

20

28.2

2.73

85

Lake

Taihu

Mixed

water**

––

––

–625

417

146

38.0

2.54

90

Microcosm

††

––

––

–1183

734

85

187.1

3.03

93

*phoDOTU

scutoff

=0.25sensu

Tanet

al.(2013).

†phoXOTU

scutoff

=0.05sensu

Iwai

etal.(2011).

‡Th

isstudy:

Lake

Alchichicaassembles:

Microbialites

=spongyan

dcolumnar

morphotype,

Bacterioplankton=dep

ths5,25an

d61m.

§OTU

sunshared

amongbacterial

assemblesan

dseasons.

¶ Seb

astian

&Ammerman

(2009):phoXfrom

theSargasso

Seaan

dChesap

eake

Bay

assemblages:seaw

ater

(dep

th=40m).

**Dai

etal.(2014):Lake

Taihuassemblages:mixed

water.

††Dai

(unpublished

):directGen

Ban

ksubmission.Lake

Taihuassemblage:

microcosm

experim

ent.

Table

2.Ph

ysicochem

ical

characterizationofLake

Alchichicalittoralzone(averageof12microbialitesamplingsites)

andwater

column(dep

ths5,25an

d61m

correspondto

water

column

bacterioplanktonsubsamples)

Dep

th

Circulation(Feb

ruary)

Stratification(August)

Littoralzone

Mean/ran

ge

5m

Epilimnion

25m

Metalim

nion

61m

Hypolim

nion

Water

column

Mean

Littoralzone

Mean/ran

ge

5m

Epilimnion

25m

Metalim

nion

61m

Hypolim

nion

Water

column

Mean

Temperature

(°C)

16.1

14.4

14.3

14.2

14.4

19.8

19.1

15.2

14.3

17.2

Conductivity(m

Scm

�1)

12.9

14.4

14.4

14.4

14.4

12.9

13.8

13.6

13.7

13.7

pH

7.6–9

.58.5

8.6

8.7

8.6

8.3–9

.58.5

8.5

8.5

8.5

DO

(mgL�

1)

6.4–1

0.1

6.6

5.9

3.9

5.7

5.9–7

.47.0

0.9

0.0

4.2

N�N

O� 3(lM)

0.66

0.19

0.67

0.53

0.49

0.59

4.85

8.04

4.32

5.92

DIN

(lM)

1.73

0.47

1.68

2.67

1.38

1.35

6.08

13.05

27.72

13.08

SRP(lM)

0.68

0.37

0.49

0.51

0.46

0.38

0.42

0.54

3.89

1.23

TP-SRP

2.3

5.8

5.8

6.5

5.9

2.4

5.6

6.2

7.7

6.3

TP(lM)

2.9

6.2

6.2

7.0

6.4

2.8

6.0

6.8

11.5

7.6

TN(lM)

38

72

64

67

69

40

65

70

82

69

DIN

:SR

P3

13

53

414

24

711

TN:TP

13

12

10

10

11

14

11

10

79



in the littoral zone, likely due to diel variability caused by

shallow depth and benthic macrophytes. Although the local

conditions for both types of microbialites varied, in general

they shared environmental conditions that distinguish the

littoral zone from the water column. In the littoral zone as

well as in the water column, nutrients, TP and TN varied

considerably between stratification and circulation, as can

be seen in Table 2.

SRP concentration was above P-limitation thresholds

(0.1 lM; Reynolds, 1999) in all sampling stations,

whereas DIN concentrations were below the N-limitation

threshold (6–7 lM; Ahlgren, 1989; Reynolds, 1999) dur-

ing circulation, and practically in the epilimnion during

the stratification. N : P ratios (DIN : SRP) were well

below 16 in most cases, pointing to the dominance of N

limitation in the Lake Alchichica water column. TN : TP

proportion also pointed to N limitation. Only during

stratification, and in particular at the 25 m depth, did the

N : P ratio suggest a condition of P limitation. In con-

trast, TN: TP ratios estimated for both microbialite types

(overall average 51 � 6; average � standard error;

spongy-type TN: TP = 54 � 7, columnar-type TN:

TP = 48 � 11) clearly indicated a strong phosphorus

limitation, marking a major functional difference between

bacterioplankton, where N-limitation is likely predomi-

nant, and microbialite assemblages, where P is limiting.

Concentrations of Ca+2, Mg+2 and Zn+2 reported for

Lake Alchichica were compared with those from seawa-

ter and Lake Taihu environmental phoX surveys (sum-

marized in Table 3). Ca+2 concentration in Alchichica is

quite low (0.27–0.37 mM), almost two orders of magni-

tude lower than Mg+2 (17.8–18.7 mM). Zn+2 concentra-

tion is high (0.459 mM, one order of magnitude above

seawater) particularly considering the salinity (8.5 g L�1)

of Lake Alchichica. The particularity of the proportions

of these metals, required by AP, and found in Lake Al-

chichica may help to widen our understanding of AP

diversity under these particular environmental condi-

tions.

Phylogenetic affiliation of phoX and phoD

A total of 218 and 180 sequences were obtained for

phoX and phoD genes, respectively. Sequences obtained

in this study were similar to AP from other environ-

mental studies and from genome databases. The

microbial consortia of Lake Alchichica (microbialites

and bacterioplankton) sequences for both phoX and

phoD were more similar between them than they were

to sequences from other environments, and were related

to Proteobacteria (mainly to Alphaproteobacteria) and

other heterotrophic groups such as Actinobacteria (Figs 3

and 4).

phoX sequences

Lake Alchichica AP phoX (from microbialites and bacte-

rioplankton) and reference sequences were organized in

five main clusters in the phylogenetic reconstruction

(Fig. 3). Cluster I (upper part of the tree) comprises

phoX-partial sequences from environmental studies along

with one sequence found only in the metalimnion of Lake

Alchichica (stratification 25 m), the single site where P

limitation was indicated by the N: P ratio. The geographi-

cal origin of the environmental sequences in Cluster I are

the Sargasso Sea surface water (Sebastian & Ammerman,

2009), and mixed water and sediments from Taihu Lake

(Dai et al., 2014). This cluster appears to be the most

unexplored phoX group, containing no sequences from

isolated strains. Cluster II comprises phoX sequences

exclusively from genome sections that belong to Betapro-

teobacteria and Actinobacteria, none of which were found

in the Alchichica microbial assemblages we studied. Clus-

ter III groups Alphaproteobacteria phosphatases or puta-

tive phosphatases and environmental phoX-partial

sequences. Most of Lake Alchichica phoX sequences

recovered from both microbialites and bacterioplankton,

were grouped here, although this clade also includes

phoX-partial sequences from marine waters and lake sedi-

ments. Alphaproteobacteria sequences in Cluster III are:

Sinorhizobium freddi (CP001389), Sinorhizobium meliloti

1021 (AL591688), Rhodobacter sphaeroides (CP000144),

Table 3. Trophic state and environmental conditions (concentration)

of major metal cofactors (Ca+2, Mg+2 and Zn+2) for bacterial AP. Data

include Lake Alchichica and environmental studies for alkaline

phosphatase gene surveys

System

Ion

Lake Alchichica

water column

Average

Sargasso Sea

Average (mM)

Lake Taihu

Mixed

water

Average Sediment

Ca+2 0.37†

0.27§10.3* 0.72¶ **

Mg+2 17.8†

18.7§53* 0.27¶ **

Zn+2 0.46‡ 0.05–9 9 10�3* 0.15¶ **

Mg : Ca 48.1†

69.3§5.30 0.38 0.88

Ca : Zn 806.1†‡

588.2§‡2 9 106 4.80 35.09

Trophic

state

Oligo-

mesotrophic

Oligotrophic Eutrophic –

*Millero (1996), †Armienta et al. (2008), ‡Mancilla-Villa et al. (2012),§Ka�zmierczak et al. (2011), and ¶Zhang et al. (2014).

Ion concentrations reported in **Wuenchuan et al. (2001) are

reported over dry mass (Ca+2 6387 mg kg�1, Mg+2 5633 mg kg�1,

Zn+2 182 mg kg�1), therefore only ionic ratios are included for com-

parison.



Ochrobactrum anthropi (CP000759), Starkeya novella

(CP002026), Mesorhizobium opportunistum (CP002279)

and Dinoroseobacter shibae DFL (CP0008830.1). Cluster

IV comprises a heterogeneous set of sequences: environ-

mental (Lake Alchichica) and genome sections from Al-

phaproteobacteria such as Paracoccus denitrificans PDI222

(CP000490.1), and members of the family Hyphomicrobia-

ceae such as Pelagibacterium halotolerans B2 (CP003075.1)

and Hyphomicrobium denitrificans, and Betaproteobacteria:

Ramlibacter tataouinensis TTB310 (CP000245) and Chlo-

roflexus aggregans (CP001337). Interestingly, most of the

sequences found in Alchichica were recovered only from

microbialites. Cluster V (bottom) is strongly separated

from the upper clades, grouping genomic phosphatase

sequences from Actinobacteria, such as Salinispora arenico-

la CNS-205 (CP000850.1), Salinispora tropica CNB-440

0.3

Alchichica lake microbialites (C)

KF891662, KF891663, KF891594

Alchichica microbialites (F)

CP001715.1 Candidatus Accumulibacter phosphatis

CP001964.1 Cellulomonas flavigena DSM 20109

KF891569, KF891574, KF891593, KF891674

Uncultured marine bacterium

30407155 Sinorhizobium meliloti 1021

KF891626, KF891635, KF891642, KF891646

CP002026.1 Starkeya novella DSM 506

KF891669

CP000830.1 Dinoroseobacter shibae DFL 12


KF891531, KF891532

CP000850.1 Salinispora arenicola CNS 205

Uncultured lake water and sediment bacterium

CP002279.1 Mesorhizobium opportunistum WSM2075

CP003563.1 Sinorhizobium fredii USDA 25

KF891519

CP001683.1 Saccharomonospora viridis DSM 43017

CP000667.1 Salinispora tropica CNB.440


Alchichica microbialites and bacterioplankton (A)

Alchichica bacterioplankton (E)

328880049 Streptomyces venezuelae ATCC 10712

CP002896.1 Amycolaptopsis mediterranei S699

KF891566

Alchichica microbialites and bacterioplankton (D)

CP002083.1 Hyphomicrobium denitrificans ATCC 51888

CP003153.1 Dechlorosoma suillum PS

CP000759.1 Ochrobactrum anthropi ATCC 49188

KF891538, KF891541, KF891559, KF891636, KF891641

CP003075.1 Pelagibacterium halotolerans B2

CP001389.1 Sinorhizobium fredii NGR 234

CP001337.1 Chloroflexus aggregans DSM 9485

Alchichica bacterioplankton


KF891585, KF891591, KF891611, KF891616, KF891617

KF891650

CP001630.1 Actinosynnema nirum DSM 43827

KF891621, KF891647


CP000144.1 Rhodobacter sphaeroides 2 4.1

CP000490.1 Paracoccus denitrificans PD1222

KF891620


KF891588

CP001867.1 Geodermatophilus obscurus DSM 43160

Alchichica microbialites and bacterioplankton (G)

15789340 Halobacterium sp. NRC.1

KF891643

CP000245.1 Ramlibacter tataounensis TTB310

Alchichica bacterioplankton (B)

751

989

997732

1000

996

908

819

1000

730

770

947

662

874

993

962

991

1000

851

1000

651

998

613

740

947

1000

996

1000

666

1000

658

840

820

991

603

603

927

569

1000

793

907

748

918

983

765

992

Cluster IV

Cluster I

Cluster II

Cluster III

Betaproteobacteria Actinobacteria

Uncultured lake (water and sediment) and marine bacterium

Actinobacteria Archaea

Alchichica microbialites and bacterioplankton

Alphaproteobacteria


Alphaproteobacteria

Fig. 3. Maximum likelihood topology for phoX alkaline phosphatase partial sequences. Magenta branches correspond to phoX sequences from

Alchichica microbialites (spongy morphological type and columnar-type) and bacterioplankton from three different depths (5, 25 and 61 m):

black branches and acquisition numbers correspond to phoX from bacterial genomes (predicted amino acid sequences domain COG3211:PhoX;

NCBI Conserved Domains) and colour branches correspond to uncultured bacterium phoX from lake water and sediments (green) and seawater

(blue). Sequences were retrieved from NCBI with BLASTN. Tree was constructed based on an alignment of c. 600 nucleotides; branches show

bootstrap testing over 1000 replicates (only bootstrap values > 60 are shown). The identity of Alchichica groups can be seen in Table S1

(Supporting Information).



(CP000667.1), Geodermatophilus obscurus DSM 43160

(CP001867.1), Streptomyces venezuelae ATCC 10712

(FR845719), Actinosynnema mirum DSM 43827

(CP001630.1) and the archaeon Halobacterium sp. NRC-1

(NC_002607). No sequences in this cluster were recov-

ered from Lake Alchichica samples.

phoD-like sequences

Microbialite and bacterioplankton phoD-like sequences

were similar to reported rhizosphere uncultured bacteria

sequences (Sakurai et al., 2008) and to genomic alkaline

phosphatase phoD-like sequences. All sequences arranged

0.2

CP000967.1 Xanthomonas oryzae pv. oryzae PXO99A

CP001001.1 Methylobacterium radiotolerans JCM 2831

CP000884.1 Delftia acidovorans

CP000304.1 Pseudomonas stutzeri A1501

CP001736.1 Kribbella flavida DSM 17836

CP001778.1 Stackebrandtia nassauensis DSM 44728

CP001644.1 Ralstonia Pickettii 12D

47118316 Bradyrhizobium japonicum

CP002738.1 Methylomonas methanica

260644157 Streptomyces scabiei 87.22

Uncultured soil bacterium

Alchichica microbialites and bacterioplankton (Z)

KF891709

KF891828, KF891857, KF891870

Uncultured rhizosphere bacterium

Alchichica microbialites and bacterioplankton (X)

CP001157.1 Azotobacter vinelandii DJ

CP001738.1 Thermomonospora curvata DSM 43183

KF891871

KF891806, KF891826, KF891845, KF891864

Alchichica microbialites and bacterioplankton (W)

KF891791, KF891809, KF891819

CP002039.1 Herbaspirillum seropedicae SmR1


CP002496.1 Pseudomonas aeruginosa M18

CP000494.1 Bradyrhizobium sp. BTAi1

gi|161727365|dbj|AB306954.1|

CP002994.1 Streptomyces violaceusniger Tu 4113

CP000633.1 Agrobacterium vitis S4

CP002735.1 Delftia sp. Cs1-4

KF891779

CP000249.1 Frankia sp. CcI3

KF891784, KF891789, KF891805, KF891823

133909243 Saccharopolyspora erythraea NRRL 2338

Alchichica microbialites (V)


KF891804, KF891725, KF891727, KF891752

CP000352.1 Cupriavidus metallidurans CH34



Alchichica microbialites (Y)



Alchichica bacterioplankton KF891866, KFKF891868, KF891876


328880048 Streptomyces venezuelae ATCC 10712


KF891728, KF891810, KF891855, KF891856

KF891757, KF891781, KF891825

CP000285.1 Chromohalobacter salexigens DSM 3043

KF891721, KF891734

CP002447.1 Mesorhizobium ciceri biovar biserrulae WSM1271

565

975

606

963

608

997

940

906

940

907

999

964

876

977

711

544

718

892

1000

841

758

614

716

911

Cluster I

Cluster II

Cluster III

Alphaproteobacteria

Alphaproteobacteria

Betaproteobacteria

Gammaproteobacteria

Actinobacteria

Alchichica lake

Alchichica lake

Alchichica lake

Alchichica lake

Alchichica lake

Alchichica lake



Fig. 4. Maximum likelihood relationships of alkaline phosphatase phoD-like based on an alignment of c. 200 nucleotides (Data S2). Partial

sequences from AP from spongy and columnar-type microbialites, and bacterioplankton from three different depths (5, 25 and 61 m), are shown

in cyan colour. Sequences from Lake Alchichica group with AP phoD-like and with sequences that correspond to the family of proteins

Metallophosphatases, also group with AP of uncultured bacteria (from rhizosphere). Black branches and acquisition numbers correspond to

Alpha-, Beta-, Gamaproteobacteria and Actinobacteria. Sequences from environmental studies are shown in different colours. Sequences were

retrieved from NCBI with BLASTN (only bootstrap values > 60 are shown). The identity of Alchichica groups can be consulted in Table S2.



in a maximum likelihood tree are shown (Fig. 4), and

grouped in three clusters (Fig. 4). In Cluster I, sequences

from both types of Alchichica microbialites found during

lake circulation grouped with uncultured bacteria

sequences and phosphatases from Actinobacteria: Strepto-

myces venezuelae (gi.328880049), Streptomyces violaceusni-

ger Tu 4113 (CP002994.1), Streptomyces scabei 87.22(gi.

260644157), Frankia sp. CcI3 (CP000249.1), Thermomo-

nospora curvata DSM 43183 (CP001738.1) and Kribella

flavida DSM 17836 (CP001736.1). Cluster II comprised a

number of sequences of bacterioplankton and some

sequences from the microbialites grouped with uncul-

tured soil bacteria and the Alphaproteobacteria: Bradyrhiz-

obium japonicum (BA000040) alkaline phosphatase.

Cluster III included most of the recovered Alchichica

phoD-partial sequences. Microbialites and bacterioplank-

ton sequences group with some AP from uncultured soil

bacteria and with proteobacterial phosphatases in general,

and in particular with Alpha-, Beta- and Gammaproteobac-

teria as follows. Alphaproteobacteria: Bradyrhizobium sp.

Btail (CP000494.1), Mesorhizobium ciceri biovar biserrulae

WSM1271 (CP002447), Agrobacterium vitis S4 (CP000633.

1), Methylobacterium radiotolerans (CP001001.1). Betapro-

teobacteria sequences (middle clade) belonging to Ralsto-

nia pickettii 12D (CP001644.1), Herbaspirillum seropedicae

SmR1 (CP002039.1) and Cupriavidus metallidurans CH34

(CP000352.1). Gammaproteobacteria sequences from Delf-

tia acidovorans (CP000884.1), Delftia sp. Cs1–4(CP002735.1), Xantomonas oryzae pv oryzae PXO99A

(CP000967.1), Azotobacter vinelandii DJ (CP001157.1),

Pseudomonas stutzeri A1501 (CP000304.1) and Pseudomo-

nas aeruginosa M18 (CP002496.1), Chromohalobacter sa-

lexigens DSM 3043 (CP000285.1) and Methylomonas

methanica (CP002738.1). Similar to phoX sequences, there

are some clades that exhibit solely microbialite or bacterio-

plankton, circulation or stratification sequences (Fig. 4).

Diversity analysis

OTU numbers for microbialites and bacterioplankton, dur-

ing circulation and stratification, were slightly higher for

phoX (OTUs = 27, similarity cutoff = 0.05, sensu Iwai

et al., 2011 for functional genes) than for phoD

(OTUs = 23, similarity cutoff = 0.25 sensu Tan et al.,

2013; Table 1). Unique sequences constituted 62.5% of

total sequences for phoX and 52.2% for phoD. OTU-based

analyses were used to explore Lake Alchichica’s intrinsic

diversity of both AP studied. Microbialites and bacterio-

plankton from Alchichica shared six phoX OTUs and 10

phoD OTUs (equivalent to 22.2% and 43.5% of total OTUs,

respectively). For both markers (phoX and phoD), micro-

bialites showed higher diversity than bacterioplankton

samples (Table 1), except for the lake stratification period,

when bacterioplankton phoX was more diverse.

An OTU-based analysis was performed with four

uncultured bacterium-phoX datasets, using sequences

based on a c. 180 amino acids alignment (Data S1). These

sets of sequences are the results of environmental studies

that followed similar methods (e.g. using the same phoX

set of degenerated primers). The sources of these data

are: seawater (Sebastian & Ammerman, 2009), ‘mixed

water’ from shallow Lake Taihu (Dai et al., 2014) and a

microcosm experiment in Lake Taihu (J.Y. Dai, Effect of

microcystis bloom decomposition on genetic diversity of

bacterial phoX in an microcosm experiment, unpublished,

GenBank direct submission). Results are heterogeneous

and summarized in Table 1. The number of phoX OTUs

found was higher in the mixed water from Lake Tai-

hu > microcosm experiment in Lake Taihu > Lake Alchi-

chica > seawater. No phoX OTUs were shared among the

three systems (even extending the cutoff to 0.10); how-

ever, groups of Lake Taihu (mixed water and microcosm

experiment) shared 34 OTUs (cutoff = 0.05). To frame

the phoX genetic potential of each assemblage, we gath-

ered relevant environmental information on these systems

(Table 3).

Discussion

Potential for DOP utilization

Warm monomictic lakes (such as Alchichica) provide a

short-term (seasonal) changing scenario for their biota,

associated to the circulation–stratification annual cycle.

Bacterial alkaline phosphatase genes (phoX and phoD-like)

were present and widespread in the microbial consortia

of this soda system, although conductivity and species

richness have often shown an inverse relationship (Wil-

liams et al., 1990). Whereas conductivity is relatively

steady, both in the water column and in the littoral zone

of Alchichica, temperature, DO and pH are more variable

in the littoral zone, where microbialites are found

(Table 2, Fig. 5). Overall, pH conditions in this system

are in the optimal activity range for AP (Jansson et al.,

1988).

The available genetic potential for DOP utilization may

be relevant for communities that face important P avail-

ability changes in the short-term (e.g. SRP concentration

in the littoral zone and in surface waters approaches P

limitation thresholds during lake stratification). A greater

diversity of AP genes was found at depths where reminer-

alization is intense, such as the base of the metalimnion

and where inorganic P was abundant (e.g. the hypolim-

netic waters, close to the sediments). The depth distribu-



tion of DOP transformation genes has been explored

recently in marine waters. Luo et al. (2011) found that

phoX and phoD genes were distributed throughout the

water column (in the surface as well as in deep waters),

whereas phoA was found exclusively in deep waters. The

bacterioplankton assemblages of Lake Alchichica had a

widespread distribution of AP along the depth and nutri-

ent gradients. This observation agrees with Luo et al.’s

(2011) observation that AP may have a variety of func-

tions, such as contributing to the degradation of refrac-

tory organic matter (Luo et al., 2011) or nucleic acids, as

was pointed out by Pinchuk et al. (2008) for extracellular

PhoX.

The high TN: TP ratios (average 51) found in micro-

bialites suggest strong P limitation within these consortia

in contrast to bacterioplankton. As a group, AP have been

reported in microbialite metagenomes; a high abundance

of these genes was found in two microbialite metage-

nomes living in the Cuatro Ci�enegas Basin, which is also

a P-scarce environment in Northern Mexico (Breitbart

et al., 2009). The expected presence and diversity of DOP

transformation genes in complex assemblages (i.e. micro-

bialites) were verified here through the vast number of

AP sequences found and the fact that bacterial AP phoX

and phoD had not been described in detail in these

microbial consortia. It is notable that over half of the

phoX identified in Alchichica were recovered only from

microbialites, suggesting there may be a higher diversity

of AP in microbialite genomes than in other assemblages

(e.g. bacterioplankton) within the same system (Table 1).

Our results highlight the relevance of heterotrophic

bacteria, particularly Proteobacteria in DOP utilization by

AP. Proteobacteria is an abundant group of Alchichica mi-

crobialite bacterial composition (Centeno et al., 2012) as

well as of bacterioplankton during the stratification per-

iod, particularly Beta- and Gammaproteobacteria (Hern�an-

dez-Avil�es et al., 2010). This result is consistent with the

studies of Sebastian & Ammerman (2009) and Luo et al.

(2009), which found a great proportion of Alphaproteo-

bacteria harbouring phoX genes in marine and estuarine

samples, and databases. Other studies have highlighted

the role of these bacteria in organic matter utilization not

only through the use of AP but also through a diversity

of extracellular enzymes (Cunha et al., 2010).

Sebastian & Ammerman (2009) found a relevant pro-

portion of AP related to Planctomycetes in their study of

oceanic water. However, we did not find AP (phoX or

phoD) sequences related to Planctomycetes, in spite of

their relevant activity in the water column of Lake Alchi-

chica during the circulation period (sensu Hern�andez-

Avil�es et al., 2010). Further studies are needed to contrast

potential vs. functional participants in DOP utilization.

The strong genetic potential for DOP utilization found in

Lake Alchichica is consistent with the tendency of the

0 2 4

SRP ( µM )

0 10

TP ( µM )

0 10

NO3– ( µM )

0 20

DIN ( µM)

0 100

TN ( µM )

0 5 10

DO (mg L–1 )

0 5 10

pH –60

–50

–40

–30

–20

–10

0 5 15

Z (m

)

T (ºC)

Lake circulation Lake stratification

Fig. 5. General landscape of Lake Alchichica and physicochemical characterization of its water column (temperature, DO, pH); nutrient

concentration of soluble reactive phosphorus (SRP), nitrate (NO�3 ), DIN; TP and TN during well established circulation (February) and stratification

(August).



lake to mesotrophy, as seen by an abundance of microor-

ganisms similar to that in systems with a higher trophic

state (Bautista-Reyes & Macek, 2012) and with the pres-

ence of seasonal blooms, as previously described (Macek

et al., 2009).

Characterization of AP found in Lake Alchichica

Although the phylogeny of bacterial AP has not been

resolved yet, there have been advances in characterization

of these enzymes recently. Zaheer et al. (2009) identified

divergence in the structure of PhoX, and proposed two

groups of PhoX differing in having a conserved glycine

(PhoX-I) or asparagine (PhoX-II) next to their putative

catalytic Ca2+ binding site. Some of the Lake Alchichica

phoX sequences grouped to Alphaproteobacteria (P. deni-

trificans and S. meliloti), considered PhoX group II (sensu

Zaheer et al., 2009). The exploration of each of these

sequences in the Conserved Domains NCBI Database and

the HMMER platform (Finn et al., 2011) showed affilia-

tion to the protein family COG3211: PhoX (predicted

phosphatase, general function prediction only;

pfam05787: DUF839). Most of the sequences were recog-

nized as bacterial proteins of unknown function

(DUF839) that contain a predicted beta-propeller repeat.

Sinorhizobium meliloti 1021(Smc02634) PhoX has a Tat

signal peptide and Ca+2 requirement similar to that of

other PhoX proteins (Roy et al., 1982). The binding Ca+2

residues were recognized in all of the phoX sequences

generated in this study using the HMMER platform

search for amino acid sequences (residues E273 and E873,

sequence 3ZWU_A, Protein Data Bank).

Our results confirm that ALPS primers (Sakurai et al.,

2008) allow successful amplification of phoD-like

sequences, findings that are shared with Tan et al. (2013).

As seen in Fig. 3, phoD from Alchichica consortia and

their predicted amino acid sequences diversity were

related to different protein domains. BLAST searches found

that microbialite and bacterioplankton sequences were

related in general to Bacillus subtilis PhoD and related

proteins, including Metallophosphatases Domain

(cd07389 MPP_PhoD; NCBI, Conserved Domains). Other

sequences recovered were related to proteins of the Metal-

lophosphatase Superfamily, Metallophosphatase Domain

(cl13995 MPP_Superfamily; NCBI, Conserved Domains),

which are partial AP sequences of uncultured bacteria

from rhizosphere samples (JN388924 and JN388926; Chh-

abra et al., 2013), and uncultured soil bacteria

(AB306951, AB306956, AB306959, AB306960, AB306963,

AB306965, AB306967; Sakurai et al., 2008); this classifica-

tion is shared with Mesorhizobium loti MAFF303099

phosphatase (BA000012, Kaneko et al., 2000) and Delftia

acetivorans SPH-1 (CP000884, Schleheck et al., 2004).

Phosphatase sequences from Bradyrhizobium japonicum

USDA 110 are also classified in the MPP_PhoD domain.

Some of these protein domains have a recent assignation

and it is likely that the affiliation of AP will be expanded

and refined in the near future, as their structural com-

plexity has been related to their substrate non-specificity

(�Strojsov�a et al., 2003; Chr�ost & Siuda, 2002), substrate

promiscuity (O’Brien & Herschlag, 2001; Lassila & Hers-

chlag, 2008) and subcellular location (Luo et al., 2009).

Amplification with degenerated primers (Sakurai et al.,

2008) was very useful to find a large set of phoD-like AP

of diverse conformations, as revealed by the current clas-

sification of protein domains (in a large set of bacterial

phyla; see NCBI, INTERPRO 37.0). The calcium-binding site

of Bacillus subtilis PhoD (residues N215 and N216; 2YEQ,

Protein Data Bank) was present in 96% of the PhoD-like

translated sequences recovered from microbialites and

bacterioplankton of Lake Alchichica.

Alkaline phosphatase diversity

Overall, AP phoX were more abundant than phoD, as

revealed by the higher number of estimated phylotypes.

Microbialites had a higher abundance and diversity of

phoX and phoD AP compared with bacterioplankton

(Table 1). A higher potential for DOP utilization through

AP in microbialites is consistent with the clear evidence

of P limitation of microbialites (TN: TP = 51) vs. a less

defined nutrient limitation of bacterioplankton, as shown

by the environmental analyses (SRP, TP, organic P and

N: P ratios, Table 2).

OTU-based analyses using partial phoX and phoD

revealed that gene pools of these AP follow different dynam-

ics along the hydrological cycle of circulation–stratification(i.e. total OTUs of bacterioplankton in the circulation per-

iod are unique). A fraction of phoX and phoDOTUs (phylo-

types) were shared between circulation and stratification,

and also between microbialites and bacterioplankton, show-

ing that the same components of the communities poten-

tially use these AP for phosphorus utilization. However, the

potential for DOP utilization seems to be harboured by dif-

ferent members of the community over time.

phoX abundance and diversity in aquatic systems

(Table 1) represent the AP phoX potential in a broad gradi-

ent of environmental features: P-status (from oligotrophic

seawater to eutrophic conditions of Lake Taihu), commu-

nity structure (bacterioplankton, microbialites and a micro-

cosm experiment) and physicochemical and geochemical

characteristics, in which we highlighted calcium,magnesium

and zinc conditions (major metal cofactors of bacterial AP).

Many of Lake Alchichica sequences were similar to

those of Alphaproteobacteria, which constitute c. 30% of

the total composition of these microbialites (Centeno



et al., 2012), but primer characteristics may be constrain-

ing these results, too, underestimating phoD (Tan et al.,

2013), as well as phoX presence and diversity. Although

phoD has been reported to be the most abundant AP in

the ocean (Luo et al., 2009) and in agricultural soils (Tan

et al., 2013), in Lake Alchichica phoX was more abundant

than phoD. This is an interesting result that should be

weighed, considering the bacterial groups that our prim-

ers are able to identify (mainly Proteobacteria).

Ionic conditions

Calcium-based AP (PhoX and PhoD-like) were diverse in

Lake Alchichica microbial communities even though the

Ca+2 concentration in this system is low in relation to

seawater (Table 3). PhoX distribution was related more

to Alphaproteobacteria (Fig. 3), and PhoD-like and AP

similar to the Metallophosphatases Superfamily of pro-

teins were more broadly distributed among bacterial

groups (Alpha-, Beta-, Gammaproteobacteria and Actino-

bacteria; Fig. 4). It is interesting that some of the Lake

Alchichica AP-partial sequences showed affiliation to

sequences from other environmental studies (marine,

estuarine and soil), whereas the metal composition (Ca+2,

Mg+2 and Zn+2) is substantially different from these sys-

tems (Table 3). Mg : Ca and Ca : Zn ratios are higher in

Lake Alchichica than in seawater. The high Mg : Ca ratio

found in Alchichica likely has a direct impact on the

composition of microbialites, which are among the few

composed mainly of hydromagnesite and hydromagne-

site-magnesite (Ka�zmierczak et al., 2011).

Conclusion

Lake Alchichica represents a variable environment in terms

of P concentration, with low N and relatively low P condi-

tions, but characterized by high Mg : Ca water ratios

relative to seawater. There is a high potential for DOP uti-

lization through microbial AP (phoX and phoD-like) in the

microbialites and the bacterioplankton. We hypothesize

that organic phosphorus is strongly utilized in Lake Alchi-

chica, which would be consistent with the presumably low

P inputs from groundwater (Ka�zmierczak et al., 2011) and

atmospheric deposition (Oseguera et al., 2010). The utili-

zation of DOP would be important to regenerate the inor-

ganic phosphorus required to sustain the relatively high

primary production during the mesotrophic stages of the

lake (Ram�ırez-Olvera et al., 2009; Macek et al., 2009). Due

to the high pH and the AP metal dependence, the condi-

tions of Lake Alchichica are potentially favourable for the

activity of AP that use Ca+2 (PhoX and PhoD), Mg+2 and

Zn+2 (PhoA) as metal cofactors. Saline alkaline systems

often exhibit unique ionic compositions and harbour

microbial communities whose metabolic potential should

be explored (sensu Antony et al., 2013). These systems may

offer an opportunity to gain insight into the elemental

dynamics related to the biogeochemistry of the planet. In

this sense, the interaction of metals that function as cofac-

tors or inhibitors of these enzymes (used for P transforma-

tions) offers an interesting study model. The role of

metallophosphatases and other P enzymes in aquatic sys-

tems is key to understanding P utilization and its link to

systems productivity, and ultimately to carbon dynamics

and plankton blooms.

Acknowledgements

This manuscript is part of the degree requirements of the

Posgrado en Ciencias del Mar y Limnolog�ıa, UNAM

(P.V.), who has a graduate student fellowship awarded by

CONACyT. We thank Luis Oseguera and the ‘Proyecto

de Investigaci�on en Limnolog�ıa Tropical’ (FES Iztacala,

UNAM) and Bernardo Valdespino for fieldwork support

in Lake Alchichica. Sergio Castillo of the Aquatic Biogeo-

chemistry Laboratory (ICMyL, UNAM) is acknowledged

for providing instruments for fieldwork and for develop-

ing chemical analyses. Additionally, we thank Osiris Ga-

ona and Antonio Cruz-Peralta for technical and

laboratory assistance. We acknowledge Marcos Merino

for the English revision of the document. Funding for this

project was granted by SEP-CONACyT No. 151796

(L.I.F.) and PAPIIT-UNAM No. IT100212-3 (L.I.F.). CO-

NACyT and UNAM funded L.I.F. with sabbatical leave

grants. All sampling was done under collector permit No.

PPF/DGOPA.033/2013 (L.I.F.).

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Table S1. Identity of phoX sequences of Lake Alchichica

arranged in a maximum likelyhood topology (Fig. 3).

Table S2. Identity of phoD sequences of Lake Alchichica

arranged in a maximum likelyhood topology (Fig. 4).

Data S1. Amino acid alignment of phoD primers with

genomic references and Alchichica sequences obtained in

this study.

Data S2. Nucleotide alignment of phoD primers with

genomic references and Alchichica sequences obtained in

this study.



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Alkaline phosphatases in microbialites and bacterioplankton from Alchichica soda lake, Mexico

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