Diversity and acyl-homoserine lactone production among subtidal biofilm-forming bacteria

Abstract

Bacteria isolated from subtidal biofilms were identified via 16S rRNA gene sequencing and screened for acyl-homoserine lactone (AHL) production. In total, 68 strains were isolated from 1 to 9 d-old subtidal biofilms developed at a coastal fish farm. Identification based on partial 16S rRNA gene sequencing revealed that these isolates were distributed among 3 phylogenetic groups (4 Bacteroidetes, 13 α-Proteobacteria and 51 γ-Proteobacteria), with most isolates belonging to the family Rhodobacteraceae and the genera Thalassomonas, Alteromonas, Pseudoalteromonas, Shewanella and Vibrio. AHL screening was performed using 2 AHL reporter strains, Agrobacterium tumefaciens A136 and Chromobacterium violaceum CV026. Results showed that 21 strains (31%) produced AHLs, including 3 Bacteroidetes, 5 α-Proteobacteria and 13 γ-Proteobacteria. All the AHL-producing α-Proteobacteria belonged to the family Rhodobacteraceae, whereas the AHL-producing γ-Proteobacteria consisted of 6 Pseudoalteromonas spp., 6 Vibrio spp. and 1 Thalassomonas sp. This is the first report of AHL-producing marine bacteria in the genera Flammeovirga, Pseudoalteromonas and Thalassomonas. The family Rhodobacteraceae (11 isolates) and the genera Vibrio (15 isolates) and Pseudoalteromonas (17 isolates) had the greatest number of AHL-producing isolates. AHL profiling of the AHL-producing isolates was performed by GC-MS. Most AHL-producing isolates produced several different AHLs, many of which were long-chain-and 3-oxo-AHLs. The widespread occurrence of AHL-producing bacteria in subtidal biofilms suggests that AHLs may play a role in the community development in this environment. KEY WORDS: Subtidal biofilm · Marine bacteria · Acyl-homoserine lactone · AHL Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 52: [185][186][187][188][189][190][191][192][193] 2008 2005). Therefore, we assume that subtidal biofilms may harbor various kinds of AHL-producing strains.Molecular mechanisms of AHL-driven bacterial QS have been well studied in the lab; however, little work has been done concerning its ecological role in the field (McLean et al. 1997, Parsek & Greenberg 2005. It had been reported that the diffusible AHLs produced by bacterial biofilms increase spore settlement of the green alga Ulva sp. (Joint et al. 2002). In a previous study, we reported the direct detection of AHLs in marine subtidal biofilms and found that AHLs affected larval behavior in the polychaete Hydroides elegans (Huang et al. 2007a). However, the diversity of AHLproducing bacteria in marine biofilms has not been shown.In the present study, bacteria were isolated from 1 to 9 d old subtidal biofilms developed in a coastal fish farm of Hong Kong, identified by partial 16S rRNA gene sequencing, and screened for AHL production. AHLs produced by bacteria were identified by gas chromatography-mass spectrometry (GC-MS). MATERIALS AND METHODSDevelopment of subtidal biofilms. Subtidal biofilms were developed from 12 to 21 October 2006 at Yung Shu...

Evidence for the dynamics of Acyl homoserine lactone and AHL-producing bacteria during subtidal biofilm formation

“…Although molecular mechanisms of AHL-regulated biofilm formation had been extensively studied in the laboratory (Davies et al, 1998;Parsek and Greenberg, 2005;Waters and Bassler, 2005); little is known about the ecological roles of AHLs in natural biofilm formation (Manefield and Turner, 2002;Parsek and Greenberg, 2005). In our parallel study, 21 AHL-producing bacteria were isolated from subtidal biofilms of different ages (Huang et al, 2008). In this study, we provided evidence that bacterial AHL signals were prevalent and variable in situ in subtidal biofilms; and the composition of AHL-producing bacteria was dynamics during biofilm development.…”

“…Relating the TRFs of AHL-producing isolates to biofilms community is in good agreement with the AHL reporter bioassay results and our isolating records (see Supplementary Table 1). For example, the three isolates sf28, sf41 and sf47, which were the only strains that induced coloration of CV026 (Huang et al, 2008), generated TRFs of 127, 204 and 312 bp that were specifically abundant in the 1-and 3-day-old biofilms (Figure 4), which also induced coloration of CV026 (Figure 1). Moreover, AHL-producing vibrios generated TRFs of 150 and 204 bp that were only dominant in the 1-day-old biofilms but not in other biofilms (Figure 4), which coincided with our isolation records that all Vibrio spp.…”

“…This is not surprising because most AHL-producing bacteria belonged to Vibrio spp., Pseudoalteromonas spp. and Rhodobacteraceae family (Huang et al, 2008) that were frequently found to be dominant in early marine biofilm communities (Acinas et al, 1999;Dang and Lovell, 2000;Lau et al, 2002;Buchan et al, 2005). The ability to use QS may be part of the genetic machinery that enables these bacteria to dominate in the subtidal biofilm community.…”

“…Analysis of TRFs in AHL-producing bacteria A total of 21 AHL-producing bacteria were isolated from the same batch of subtidal biofilms in a parallel study (Huang et al, 2008) and subjected to TRFLP analysis. Genomic DNA was extracted from pure colonies of each isolate using a bacterial genomic DNA extraction kit (TaKaRa, China), whereas the primers, restriction enzyme and PCR conditions to obtain TRFs from each isolate were the same as mentioned above.…”

Pedobacter xixiisoli sp. nov., isolated from bank soil

“…The 16S rRNA gene of strain S27 T was amplified using universal primers 27F and 1492R as described by Huang et al (2008). The purified PCR product was cloned into the pMD19-T vector and the recombinant plasmid was then transformed into Escherichia coli DH5a according to the manufacturer's instructions (TaKaRa).…”