Cyanobacteria from Brazilian building walls are distant relatives of aquatic genera

Cyanobacteria from Brazilian building walls are distant relatives of aquatic genera.

Peter M. Gaylarde1, Cesar A.Crispim2, Brett A. Neilan3, Christine C. Gaylarde4*

1Porto Alegre, Brazil

2Curso de Pos-graduacao em Microbiologia Agricols e do Ambiente, Universidade

Federal do Rio Grande do Sul (UFRGS), Brazil

3University of New South Wales, Sydney, Australia

4Dept. Biophysics, UFRGS, Porto Alegre, 91000-970, Brazil.

Tel/Fax (+55) 51 336 6029

E-mail [email protected] *corresponding author

Running title: Cyanobacteria on Brazilian buildings

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Abstract

The 16S rDNA from 22 cyanobacteria isolated from biofilms on walls of modern and

historic buildings in Brazil was partially sequenced (~350 bp) using specific primers.

The cyanobacteria with the closest matching sequences were found using the BLAST

tool. The sequences were combined with 52 other cyanobacterial sequences already

deposited in public data banks and a dendrogram constructed, after deletion from each

sequence of one of the variable 16S rDNA regions (VI). The newly sequenced

organisms fitted well within their respective families, but their similarities to other

members of the groups were generally low, less than 96%. Close matches were found

only with one other terrestrial (hot dry desert) cyanobacterium, Microcoleus sociatus,

and with Anabaena variabilis. Phylogenetic analysis suggested that the deletion of the

hypervariable regions in the RNA structure is essential for meaningful evolutionary

studies. The results support the standard phylogenetic tree based on morphology, but

suggest that these terrestrial cyanobacteria are distant relatives of their equivalent

aquatic genera and are, indeed, a distinct population.



INTRODUCTION

Phototrophic microorganisms growing on modern and historic buildings cause

deterioration of the structure (Gaylarde and Gaylarde, 2004b). A large number of

genera are present, and these grow in biofilms (microbial consortia) on stone and

painted surfaces (Anagnostidis et al., 1983; Ortega-Calvo et al., 1995; Gaylarde and

Gaylarde, 2000).

In Latin America, the major biomass on these building surfaces is composed of

cyanobacteria (Gaylarde and Gaylarde, 2004a). They lead to discoloration (aesthetic

deterioration), as well as degradation of the surface (Warscheid et al., 1991; Ortega-

Calvo et al., 1995). Cyanobacteria can survive repeated drying and rehydration cycles

(Whitton, 1992) and high UV levels (Garcia-Pichel et al., 1992; Matsunaga et al.,

1993; Chazal and Smith, 1994) and for these reasons they are particularly important

organisms in biofilms on exposed surfaces in Latin America.

Cyanobacteria (previously known as blue-green algae) are prokaryotic photosynthetic

microorganisms, which are found worldwide. They have long been recognised as

important soil and water organisms, where their activities of nitrogen fixation and

toxin production are of special interest. Only more recently has their role in the

deterioration of built structures been studied.

Traditional methods for the detection of cyanobacteria are based on their culture on

specific growth media. These isolation techniques were developed in the area of

aquatic microbiology and later extended to terrestrial habitats (soil). They involve

enrichment in liquid media, followed by isolation by micromanipulation or culture on

solid medium (Rippka et al., 1979). However, these methods can result in the

detection of artificially low numbers and diversity, due, in part, to the presence of

inhibitory and predatory organisms, such as fungi, bacteria, and protozoa (Gaylarde



and Gaylarde 2000). Many cyanobacterial species from dry environments are lost in

culture because of the activity of browsing and inhibitory organisms, and, of course,

many microorganisms present in the environment are not detected by current culture

techniques (Ward et al., 1990).

Although 16S rRNA gene sequencing has been used to study the relationship between

cyanobacteria (Wilmotte et al., 1993; Nelissen et al., 1996; Turner, 1997), there has

been practically no work on the molecular biology of cyanobacteria on buildings.

Tomaselli et al. (2000) used amplified ribosomal DNA restriction analysis (ARDRA)

on axenic cyanobacterial isolates from Italian monuments and constructed a

dendrogram that provided some idea of biofilm diversity. This approach did not,

however, overcome the problem of selective culture of organisms from a mixed

population. Crispim et al. (2003) and Gaylarde et al. (2004) described a method to

identify cyanobacteria in mixed biofilms on external walls of historic buildings that

does not rely on isolation and this is the approach used here to examine the

phylogenic relationships between these biofilm and other cyanobacteria.

MATERIALS AND METHODS

Sampling and microbiological analysis. Samples of biofilms were taken from the

external surfaces of modern and historic buildings in Porto Alegre, Ouro Preto,

Tiradentes and São Paulo, Brazil, using the adhesive tape method (Gaylarde and

Gaylarde, 1998; Shirakawa et al., 2002). All surfaces showed visible discoloration,

generally grey/black, green, or orange/red in appearance.

For microbiological analysis, tape samples were placed directly on solid Modified

Knop’s Medium (MKM; Gaylarde and Gaylarde, 2004) and incubated at 25oC in an

illuminated BOD incubator. Some organisms were isolated in liquid MKM by

repeated subculture after an initial growth period on solid medium.



Identification. Microorganisms in rehydrated biofilms (4h after contact with MKM)

and in culture were identified by standard morphological methods (Holt et al., 1994;

Castenholz, 2001). Cyanobacteria were also identified by molecular techniques, as

briefly described in the next sections (Gaylarde et al., 2004).

DNA extraction. Genomic DNA was extracted from some cyanobacterial isolates

using lysozyme/proteinase K/SDS for lysis, followed by a phenol/chloroform/isoamyl

alcohol extraction (Gaylarde et al., 2004). Extracted DNA was amplified using the

PCR parameters described below.

DNA amplification For the “direct” PCR, microcolonies or filaments from the

rehydrated, or briefly incubated, biofilms and well separated filaments isolated from

liquid medium were placed in 10 µl of TE in an Eppendorf and freeze-thawed five

times in the freezer compartment of a domestic refrigerator (-18oC) and at room

temperature (23oC). After the final thawing, dNTPs, primers, MgCl2, buffer and Taq

DNA polymerase were added directly in the appropriate amounts (Gaylarde et al.,

2004) to the Eppendorf to give a final volume of 25 µl. The PCR reaction was carried

out in an Applied Biosystems GeneAmp System 2400 (PE Applied Biosystems,

Fullerton, CA) with the following programme: 92oC for 2 min, 30 cycles of 92oC for

20 s, 55oC for 30 s, 72oC for 50 s and a final extension at 72oC for 2 min. The

products were visualized by electrophoresis in 1% agarose gels using ethidium

bromide staining.

Primers. 16S rRNA primers designed by Neilan et al. (1997, 2002) were used. The

forward primer, 27F1, is a universal bacterial primer with the sequence

5'-AGAGTTTGATCCTGGCTCAG-3', based on primer 27F (Giovannoni, 1991) and

is identical to A2 (Iteman et al., 1995). The reverse primer, 408R, is specific for

cyanobacteria and has the sequence 5'-TTACAAYCCAARRRRCCTTCCTCCC-3'.



These primers produce amplified fragments of around 400 bp. Primers were used at a

concentration of 10 pmol/25µl PCR reaction.

DNA sequencing. Purified PCR products were sequenced with the reverse primer

408R using Big Dye terminator technology (PE Applied Biosystems) and the

sequencing gels were run and analysed by the University of New South Wales

Genomic Analysis Facility. In some cases, both primers were used separately for

sequencing. The consensus sequences were submitted to the BLAST search tool

(http://www.ncbi.nlm.nih.gov/BLAST) and nearest matches and those conforming to

the morphological appearance of the cells were recorded. Dendrograms were

constructed for the sequences, along with others of similar groups selected randomly

from public databases, using the CLUSTAL-X program (Higgins et al., 1992) and

bootstrap with 1000 resampling events. An unrooted tree was constructed using a

heuristic approach (Swofford et al., 1996). Close inspection of the sequences revealed

that the hypervariable region, VI, differed not only in sequence but also in length and

interfered with the CLUSTAL alignment. This section was therefore removed, the

dendrogram reconstructed and the BLAST analyses repeated using these modified

sequences.

RESULTS AND DISCUSSION

The cyanobacteria from our samples, and their similarities with public database

organisms, are shown in Table 1. The results are shown with and without the

sequence before base 101 (E. coli position), which includes VI (see Figure 1).

Analysis of the secondary structure indicated that the bases that did not match

deposited sequences were concentrated in variable region I. Since the alignments

performed by the CLUSTAL-X program failed to retain the complementarity of the

sequences where differences in length of VI occurred, the subsequent dendrogram was



determined by bootstrapping after deletion of VI and the preceding part of the

sequence. The dendrogram is shown in Figure 2. The percentage match of sequences

with those of other organisms in the public databases increased in all cases after this

modification.

Our results demonstrate that the removal of the VI region of the genome improved the

similarity levels of the new sequences to those in public databases. Some workers

(Stackebrandt et al., 1991; Heuer et al., 1999) have suggested that phylogenetic

studies should be carried out on partial sequences of the 16S rRNA variable regions,

since they represent areas where evolutionary change may take place in an otherwise

exceptionally conserved genome. Others, however, recommend the removal of the

variable regions after their identification with dedicated programs (Crosbie et al.,

2003).

Cyanobacteria are notable for the short length of their 16S rDNA genome compared

to other eubacterial groups; substantial deletions between E. coli positions 68 to 101,

197 to 220 and 452 to 480 are consistently present. However, it is only in the case of

region 68 to 101 (VI) that the length of the deletion is variable (Figures 3a-c). The

length of VI does not vary systematically between aquatic and terrestrial

cyanobacteria; however, several of the terrestrial organisms have very distinct

sequences in this region, not found in related, but non-terrestrial, genera. We suggest

that variable regions, if they have any utility, should be used only for phylogenic

studies at the genus level, especially since alignment methods do not work for regions

of variable length.

The dendrogram produced from the modified sequences (Figure 2) shows that the 16S

rRNA-based phylogeny is broadly in agreement with accepted phylogenies based on

morphology and that the cyanobacteria from buildings group well with their aquatic



neighbours at the family or genus level. The small number of organisms of subgroup I

(Chroococcales) are scattered throughout the diagram, showing that morphological

taxonomy is inadequate to treat this group realistically. Turner (1997) stated that the

genus Synechococcus is polyphyletic, emphasising this point.

Epilithic cyanobacteria (including those from painted stone surfaces) must be well

adapted to survive frequent desiccation, high salt levels and extremes of temperature.

The epilithic environment must be considered as a very distinct ecological niche.

Terrestrial cyanobacteria from painted or unpainted stone surfaces in Brazil form a

distinct population that differs from the better-studied aquatic members of this group.

Major differences include the fact that cyanobacteria found on solid surfaces include

no members of genera in which the production of gas vacuoles is considered a

fundamental character and that in the tropics (or at altitude) the cells frequently have

thick sheaths, which, like the cell cytoplasm, may be heavily pigmented. Figure 4(a-

d), in the web-based supplementary material, shows this feature, as well as the range

of morphological variation of Scytonema spp. ccg16, ccg25 and ccg26, which show a

very close relationship and cluster tightly in the dendrogram. These Scytonema spp.

differ from other environmental organisms that cluster with Scytonema hofmannii in

that dark pigments are present in the cell cytoplasm, false branches are more common

and consistently geminate and on media rich in nitrogen heterocysts are frequent. A

thick, pigmented sheath is normal in both groups in the environment and this usually

becomes thin and colourless in culture.

A similar grouping, with a tight cluster of terrestrial strains of Nostoc, is seen in the

dendrogram; these sequences are rooted in the same branch as deposited sequences of

Nostoc, Anabaena and Nodularia, but are clearly distant.



One of our isolates most closely matched the M. sociatus 16S rDNA short sequence

deposited by Garcia-Pichel et al. (2001); however it was identified morphologically as

Plectonema (Table 1). Boyer et al. (2003) studied 31 isolates of Microcoleus from

desert crusts and found the M. vaginatus clade was most similar to published

sequences from Trichodesmium and Arthrospira. They concluded that the genus

requires revision and our result emphasises this. The M. vaginatus morphotype

isolated from terrestrial environments must be regarded as a very diverse group with a

highly conserved 16S rDNA sequence. This analysis once again highlights the

problems of attempting to use morphological features for phylogenic analysis, since

organisms with very close 16S rDNA sequences may have very different appearances.

Almost all of the cyanobacteria show changes in morphology in response to

environmental conditions, and in the case of the heterocystous cyanobacteria and the

baeocyte-forming groups, this morphological variation is always large. We recognise

Plectonema as any filamentous, non-heterocystous cyanobacterium, growing within a

sheath, which produces false branches at some stage of growth. Since the branching

character includes a wide range of stable morphotypes, defined by the width and

length of the cells, their shape and colour, this is almost certain to represent several

genera, which will be phylogenically distant and, indeed, the existence of the genus

Plectonema has been questioned (Castenholz, 2001).

The dendrogram shows that these terrestrial cyanobacteria fit well to the taxonomic

positions of related genera, but also shows that their genetic distance from other

members of their morphologically identified genera is large. The table confirms this

result. The physiological adaptations for survival in the dry environment of terrestrial

surfaces would lead one to expect the emergence of novel taxa. The ribosomal



genome of terrestrial cyanobacteria is distinct from those of related aquatic genera and

this indicates prolonged isolation of the two groups.

Acknowledgements

We wish to thank the Brazilian agency CNPq for funding for materials and a

postgraduate grant to CAC. BAN thanks the Australian Research Council for financial

support.



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Legends for figures

Figure 1

The structure of E. coli 16S rRNA from base 28 to base 408 is shown with variable

regions VI and VII marked. The simplified structure is modified from Cannone et al.,

(2002), in such a way that the E. coli 16S rRNA will show variation with respect to

cyanobacteria; the modification has been made with reference to all bacterial

sequences. Suzuki et al. (1998) state that VI runs from 72 to 101, inclusive, whereas

our observations show that it runs from 69 to 100 in all cyanobacteria. The pairing

between bases at positions 69 and 99 is generally conserved in cyanobacteria.

VII runs from 176 to 221 (Suzuki et al., 1998). Two helical regions, denominated

Helix 1 (136 to 142 and 221 to 227, inclusive) and Helix 2 (154 to 156 and 165 to

167, inclusive), show marked variability, in which, nevertheless, complementarity is

highly conserved. Within VII, bases 195 to 197 are conserved in all the bacteria from

deposited sequences that we have examined; this region is frequently complimentary

to 180-182 in cyanobacteria. Two regions, VIIa and VIIb, show features characteristic

of cyanobacteria. VIIa runs from 183 to 193 and its variability is very large. VIIb (198

to 205 and 214 to 219) is deleted in cyanobacteria. This same deletion is shared with

bacteria of the genus Clostridium and its length varies in other bacterial genera.

Wide bars show canonical pairing (G:C and T:U), narrow bars show G:U pairs and

dots mark other non-canonical pairs which contribute to the secondary structure.

Lines mark the E. coli positions, which are numbered at every tenth base.



Figure 2

Dendrogram constructed from sequences starting at E. coli position 101. The position

of Leptolyngbya sp. ccg40 is marked to indicate that this short sequence clusters with

its nearest relatives, but that the genetic distance is increased because of the short

sequence used to give the match. This indicates that bootstrapping is robust for

sequences of variable length, but shows that the degree of relatedness cannot be

estimated when such variation is inherent. In the coloured diagram (supplementary

web material), black = subgroup I, red = subgroup II, blue = subgroup III and green =

heterocystous cyanobacteria. The colours group well together, even though the stems

of the tree do not always give the same message.

Figure 3

This figure shows the variation in length of VI in cyanobacteria. The diagrams show

that when helical regions are present they are well paired.

3a. Nostoc ccg19, showing a VI helix of typical cyanobacterial length.

3b. A Phormidium murrayii sequence, showing minimum sequence length

3c. The longest VI helix found, Nostochopsis lobatus.

Figure 4 (Coloured photos available in supplementary Web material)

Photomicrographs showing Scytonema ccg25 (4a), Scytonema ccg16 (4b) and

Scytonema ccg26 (4c and d), all grown on MKM. The bar markers are 10µm.



Caption for Table 1

This summarises the morphological identity of the organisms sequenced and the

nearest matches of the sequences to deposited organisms in the BLAST database. The

nearest matches to the morphological identifications are also given. The similarities

are for the full sequence and for the short sequence from E. coli position 101 onward,

together with the percentage similarities with and without the sequence preceding

base 101.



Figure 1



Figure 2



Figure 3 (a)

(b)

(c)



Figure 4

(a) (b)

(c)

(d)



Table 1 Code Morphological Town Nearest match(es) Short sequence Full sequence Difference between Percentage match identity Matches Mismatches Matches Mismatches mismatches Short Full ccg08 Lyngbya POA Phormidium murrayii 253 11 282 17 6 95.8 94.3 ccg10 Nostoc OP Nostoc sp. PCC 9229 254 8 303 15 7 96.9 95.3 ccg13 Nostoc OP Nostoc sp. 255 7 308 16 9 97.3 95.1 ccg 16 Scytonema POA Mastigocladopsis repens MORA 246 16 n.a. n.a. n.a. 93.9 n.a. Nostoc sp. 8941 244 18 296 23 5 93.1 92.8 Scytonema sp. IAM M-262 240 22 292 27 5 91.6 91.5 ccg 19 Nostoc SP Anabaena variabilis 251 2 305 3 1 99.2 99.0 Nostoc sp. PCC 7120 247 6 299 11 5 97.6 96.5 ccg 23 Tolypothrix POA Nostoc sp. PCC 7120 259 13 310 19 6 95.2 94.2 Tolypothrix sp. IAM M-259 250 22 301 28 6 91.9 91.5 ccg 24 Tolypothrix POA Nostoc sp. PCC 7120 249 13 300 19 6 95.0 94.0 Tolypothrix sp. IAM M-259 240 22 291 28 6 91.6 91.2 ccg 25 Scytonema POA Mastigocladopsis repens MORA 243 13 n.a. n.a. n.a. 93.8 n.a. Nostoc sp. 8941 241 18 293 23 5 93.1 92.7 Scytonema sp. IAM M-262 237 22 289 27 5 91.5 91.5 ccg 26 Scytonema POA Mastigocladopsis repens MORA 257 14 n.a. n.a. n.a. 94.8 n.a. Nostoc sp. 8941 255 16 307 21 5 94.1 93.6 Scytonema sp. IAM M-262 251 20 303 25 5 92.6 92.4 ccg 28 Lyngbya POA Leptolyngbya sp. PCC 73110 208 22 258 29 7 90.4 89.9 L. foveolarum 207 23 257 30 7 90.0 89.5 ccg 29 Plectonema POA Microcoleus sociatus MPI 96MS.KID 270 13 n.a. n.a. n.a. 98.9 n.a. Phormidium sp. ETS-05 266 16 316 23 7 94.3 93.2 ccg 32 Plectonema POA Plectonema boryanum 225 22 276 28 6 91.1 90.8 ccg 34 Leptolyngbya POA Leptolyngbya sp. 252 8 308 11 3 96.9 96.6 ccg 38 Lyngbya POA Phormidium murrayii 241 10 272 14 4 96.0 95.1 Symphyonema sp. 1269-1 229 22 278 31 9 91.2 90.0 ccg 40 Leptolyngbya OP Leptolyngbya PCC7104 231 12 244 14 2 95.1 94.6 ccg 44 Tolypothrix POA Cyanospira rippkae 251 12 303 18 6 95.4 94.4 Tolypothrix sp. IAM M-259 243 20 294 26 6 92.4 91.9 ccg 46 Subsection II T Chroococcidiopsis sp. BB96.1 246 17 n.a. n.a. n.a. 93.5 n.a. Chroococcidiopsis sp. BB79.2 245 19 297 24 5 92.8 92.5 ccg 48 Chlorogloeopsis OP Nostoc sp. 'Azolla cyanobiont' 268 12 319 18 6 95.7 94.7 Chlorogloeopsis sp. PCC 6718 259 21 310 27 6 92.5 92.0 ccg 49 Nostoc OP Nostoc sp. PCC 9229 251 3 297 12 9 98.8 96.1 ccg 50 Chlorogloeopsis OP Nostoc sp. 'Azolla cyanobiont' 268 13 319 19 6 95.4 94.4 Chlorogloeopsis sp. PCC 6718 259 22 310 28 6 92.2 91.7 ccg 51 Leptolyngbya OP Leptolyngbya PCC7104 268 7 321 9 2 97.5 97.3 ccg 52 Leptolyngbya OP Leptolyngbya PCC7104 263 11 315 14 3 96.0 95.7 POA = Porto Alegre; OP = Ouro Preto; SP = Sao Paulo; T = Tiradentes; n.a. = not available (short sequence deposited in data bank)



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Cyanobacteria from Brazilian building walls are distant relatives of aquatic genera