Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable - PubMed (original) (raw)

Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable

D G Burns et al. Appl Environ Microbiol. 2004 Sep.

Abstract

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10(6) CFU/ml) were obtained on solid media. Long incubation times (> or =8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.

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Figures

FIG. 1.

FIG. 1.

Viable counts at 12 weeks on six different culture media. Samples of crystallizer pond water were serially diluted, plated on solid media, and incubated at 37°C for 12 weeks, and the colonies were then counted as described in Materials and Methods. Bars representing counts at three dilutions, 10−3 (black), 10−4 (gray), and 10−5 (dotted), of each medium are shown. Error bars represent 1 standard deviation.

FIG. 2.

FIG. 2.

Viable counts on three different solid media versus incubation time. Samples of crystallizer pond water were serially diluted, plated on solid media, and incubated at 37°C, and the colonies were counted at 3, 8, and 12 weeks as described in Materials and Methods. Error bars represent 1 standard deviation. Plots represent the viable counts on MGM (triangles), organic acids (circles), and 0.01% NB (squares).

FIG. 3.

FIG. 3.

Cumulative isolate diversity versus incubation time. Colonies from the different media were selected from plates at 3, 8, and 12 weeks, examined by 16S rRNA gene sequencing, and grouped according to sequence similarity (see text for details). The labels denote the different groups as follows: Hfx., Haloferax group; Hrr. 1, Halorubrum group 1; Hrr. 2, Halorubrum group 2; ADL, ADL group; Nnm., Natronomonas group; other, other organisms.

FIG. 4.

FIG. 4.

Diversity of isolates and sequence library sequences. The bar chart compares the 16S rRNA sequence diversities of 57 sequenced clones from the sequence library (right) and of 66 sequenced isolates (left). The labels denote the various sequence groups recovered, as follows: Hfx., Haloferax group; Hrr. 1, Halorubrum group 1; Hrr. 2, Halorubrum group 2; ADL, ADL group; Nnm., Natronomonas group; SHOW, SHOW group; other, other organisms.

FIG. 5.

FIG. 5.

Phylogenetic tree reconstruction for 16S rRNA sequences of isolates and cloned genes recovered from a crystallizer pond. Isolates from the present study all have the prefix CSW and are shown in bold. Cloned sequences are not in bold and have the prefix CSWMA or CSWFA. Filled triangles indicate clades, with the vertical height being proportional to the numbers of sequences (also indicated to the right) and with the right side diagonal reflecting the shortest and longest branch lengths within the clade. Significant bootstrap values (≥75%; 1,000 replicates) are indicated by filled circles at branch points. Scale bar = 0.1 expected nucleotide substitutions per site. The tree was rooted by using Methanospirillum hungatei DSM 864T (accession no. M60880) as the outgroup (not shown). Sequences from validly published species are designated by their species names and culture collection accession numbers. Sequences of nonvalidly described isolates are indicated by their strain names (T1.3 and Nh.2) and their database accession numbers. A previously published clone sequence of an uncultivated square haloarchaeon (SHOW group) is denoted by its accession number, X84084.

FIG. 6.

FIG. 6.

Phylogenetic tree reconstruction for the ADL group, including 16S rRNA sequences of both cloned sequences (CSWFA or CSWMA prefix) and isolates (CSW prefix and bold type). The tree reconstruction is described in Materials and Methods, while the details of the isolates, cloned sequences, and bootstrap significance are the same as those described for Fig. 5. The outgroup sequences (not shown) were those of Haloferax volcanii NCMB 2012T and Halogeometricum borinquense ATCC 700274T (others were tested but did not alter the topology). Scale bar = 0.01 expected nucleotide substitutions per site.

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