Characterization of an intertidal cyanobacterium that constitutes a separate clade together with thermophilic strains

Figures & data

Table 1. Physico-chemical parameters of water samples from inshore and intertidal pools at Luz beach, Portugal.

Parameters (units)	Samples
	Seawater	Puddle 1	Puddle 2^b
Temperature (°C)^a	18	24.8	22.3
pH^b	8.2	8.5	8.9
Salinity (‰)^a	33.3	32.5	4.1
Conductivity (µS cm^−1)	45 500	40 800	6640
Chloride (mg l^−1)	20 000	18 000	2200
Sulfate (mg l^−1)	2600	2200	283
Total alkalinity (mg l^−1 CaCO3)	104	118	49
Bicarbonate (mg l^−1)	127	144	23
Iron (µg l^−1)	<50	<50	<50
Nitrate (mg l^−1)	2.5	5.6	3.2
Nitrite (mg l^−1)	<0.03	<0.03	<0.03
Ammonium (mg l^−1)	<0.05	<0.05	0.1
Total organic carbon (mg l^−1)	<1	6	3.6
Sodium (mg l^−1)	12 000	10 000	1400
Calcium (mg l^−1)	402	385	54
Magnesium (mg l^−1)	1400	1200	141
Potassium (mg l^−1)	425	415	54
Phosphorus (mg l^−1 P2O5)	0.05	0.05	0.04
^aMeasurements in situ using the multi-parameter field meter MultiLine P3 pH/LF SET (WTW GmbH, Weilheim, Germany). Air temperature was 19°C.
^bDuring low tide some puddles get an input of groundwater, changing their physico-chemical parameters.

Table 2. Target genes/sequences and oligonucleotide primers used in this study.

Target gene/sequence	Primer pair^a	Sequence^b 5′ → 3′	Expected size (bp)	Reference
16S rRNA	8-27F	AGAGTTTGATCCTGGCTCAG	773	Weisburg et al., 1991
	CYA781R (A)	GACTACTGGGGTATCTAATCCCATT	1133	Nübel et al., 1997
	CYA781R (B)	GACTACAGGGGTATCTAATCCCTTT		Nübel et al., 1997
	CYA359F (A)	GGGGAATCTTCCGCAATGGG		Nübel et al., 1997
	CYA359F (B)	GGGGAATTTTCCGCAATGGG		Nübel et al., 1997
	1492R	GGTTACCTTGTTACGACTT		Weisburg et al., 1991
rRNA ITS: ITSc	CSIF	GYCACGCCCGAAGTCRTTAC	450–900	Janse et al., 2004
	ULR	CCTCTGTGTGCCTAGGTATC
23S rRNA	ULF	GATACCTAGGCACACAGAGG	1001	This work
	23S1027R	GCTGGTGGTCTGGGCTGTTT	1133	This work
	23S800F	GGTTAGGGGTGAAATGCCAA	781	This work
	23S1932R	CCTTAGGACCGTTATAGTTA		Sherwood & Presting, 2007
	23S1682F	CTCTCTAAGGAACTCGGCAAA
	p23SrV_r1	TCAGCCTGTTATCCCTAGAG
rpoB	rpoBF^c	GTAGTTGTARCCNTCCCA	520–635	Rajaniemi et al., 2005
	rpoBR^c	RCMGCMGACGAAGAAGACG
cpcB-IGS-cpcA	PCβF	GGCTGCTTGTTTACGCGACA	500–740	Neilan et al., 1995
	PCαR	CCAGTACCACCAGCAACTAA
nifH	nifH4	TTYTAYGGNAARGGNGG	361	Omoregie et al., 2004
	nifH3	ATRTTRTTNGCNGCRTA
	nifH2	ADNGCCATCATYTCNCC
	nifH1	TGYGAYCCNAARGCNGA
nifK	nifK0F (A)	CAAGGTTCTCAAGGTTGCGTT	1139	This work
	nifK1F (B)	CAAGGTTCTCAAGGTTGTGTG
	nifK4R	TGTAAGTGGTGGCGATCAAAGA
^aF (forward) and R (reverse); (A) and (B) - primers used together in a mixture with equimolar concentration.
^bDegenerate primers: D = A, G or T; M = A or C; R = A or G; Y = C or T; N = A, C, G, or T.
^cAlthough these primers are published as shown here, primer F is antisense and primer R is sense.

Figs 1–8. Sampling site and samples of the colonial cyanobacterium LEGE 06123. Figs 1, 2. The sampling site at Luz beach, showing location in southern Portugal (arrowhead). Fig. 3. Unicellular cyanobacterium (arrowhead) in mat collected from shallow puddle. Fig. 4. Isolate LEGE 06123 showing sub-spherical cells which divide irregularly in various planes with dispersion through disintegration of colonies after intensive cell division (arrow). Fig. 5. Firm sheath (arrow) and amorphous polysaccharide layer (arrowhead) surround cells stained with Alcian blue. Figs 6–8. Ultrastructure (TEM) showing the laminated nature of the sheath and the concentric arrangement of thylakoids which can exhibit invaginations towards the centre of the cell (sh, sheath, th, thylakoids). Scale bars: 10 µm (light micrographs); 1 µm (TEM).

Table 3. Morphometric measurements of cyanobacterial strain LEGE 06123.^a

Stage	Cell		Sheath (µm)
	Length (µm)	Width (µm)
Single cell	4.3 ± 0.8	3.6 ± 0.7	0.62 ± 0.15
Two cells	4.1 ± 1.1	2.7 ± 0.6	–
Four cells	3.2 ± 0.6	2.8 ± 0.4	–
Colonies	3.2 ± 0.9	2.7 ± 0.6	–
	Ø colony (µm) 12.02 ± 2.19
^aValues are means ± SD (n = 10, n = 15 for colony diameter).

Fig. 9. Growth curves of LEGE 06123 in ASNIII medium at various temperatures.

Fig. 10. Growth curves of LEGE 06123 in modified ASNIII medium with various NaCl concentrations.

Fig. 11. Growth curves of LEGE 06123 in ASNIII medium supplemented with various concentrations of iron (FeCl₃· 6H₂O).

Fig. 12. Transmission electron micrograph of a 14-day-old culture of the cyanobacterium LEGE 06123 in liquid medium ASNIII supplemented with 400 µM of iron (FeCl₃· 6H₂O). The arrow shows the metal accumulation outside the cell. Scale bar: 0.5 µm.

Fig. 13. Phylogenetic tree of 16S rRNA gene sequences from several cyanobacteria generated by NJ analysis. LEGE 06123 is shown in bold. The tree was rooted with the outgroup Chloroflexus aurantiacus J-10-fl (D38365.1), which was later pruned from the tree. Bootstrap values >50% are indicated beside the nodes. Each taxon is identified by the accession number (brackets) and whenever possible by the name.

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