Contemporary microbial community analysis frequently involves PCR-amplified sequences of the 16S rRNA gene (rDNA). However, this technology carries the inherent problem of heterogeneity between copies of the 16S rDNA in many species. As an alternative to 16S rDNA sequences in community analysis, we employed the gene for the RNA polymerase beta subunit (rpoB), which appears to exist in one copy only in bacteria. In the present study, the frequency of 16S rDNA heterogeneity in bacteria isolated from the marine environment was assessed using bacterial isolates from the red alga Delisea pulchra and from the surface of a marine rock. Ten strains commonly used in our laboratory were also assessed for the degree of heterogeneity between the copies of 16S rDNA and were used to illustrate the effect of this heterogeneity on microbial community pattern analysis. The rock isolates and the laboratory strains were also used to confirm nonheterogeneity of rpoB, as well as to investigate the versatility of the primers. In addition, a comparison between 16S rDNA and rpoB PCR-DGGE (denaturing gradient gel electrophoresis)-based community analyses was performed using a DNA mixture of nine isolates from D. pulchra. Eight out of 14 isolates from D. pulchra, all rock isolates, and 6 of 10 laboratory strains displayed multiple bands for 16S rDNA when analyzed by DGGE. There was no indication of heterogeneity for either the rock isolates or the laboratory strains when rpoB was used for PCR-DGGE analysis. Microbial community pattern analysis using 16S rDNA PCR-DGGE showed an overestimation of the number of laboratory strains in the sample, while some strains were not represented. Therefore, the 16S rDNA PCR-DGGE-based community analysis was proven to be severely limited by 16S rDNA heterogeneity. The mixture of isolates from D. pulchra proved to be more accurately described using rpoB, compared to the 16S rDNA-based PCR-DGGE.

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We report for the first time the production of acyl homoserine lactones (AHLs) by bacteria associated with marine sponges. Given the involvement of AHLs in bacterial colonization of many higher organisms, we speculate that such quorum sensing signals could play a part in interactions between sponges and the dense bacterial communities living within them.Many gram-negative bacteria utilize acyl homoserine lactone (AHL)-mediated signaling systems to communicate with one another (9,27,33). These systems involve the production of low-molecular-weight molecules that accumulate with increasing bacterial numbers and thus provide an index of population density. When a threshold bacterial density (and corresponding AHL concentration) is reached, AHLs interact with transcriptional activators to trigger the expression of target genes. Many terrestrial bacteria produce AHLs, yet, beyond the well-characterized Vibrio fischeri-squid symbioses (3, 22, 24), relatively little is known about the occurrence of AHLs in marine environments. The recent finding that bacteria in marine snow are capable of producing AHLs (12) suggests that such signaling molecules could be widespread. In other studies, the addition of AHLs and cyclic AMP (4) or siderophores (14) to marine growth media increased bacterial culturability and growth, respectively.Many AHL-regulated phenotypes are involved in bacterial colonization of higher organisms, e.g., bioluminescence in V. fischeri (22,24) and conjugal plasmid transfer in Agrobacterium tumefaciens (23,36). Given the dense bacterial communities associated with marine eukaryotes such as sponges (8, 34, 35), macroalgae (18, 20), or ascidians (29, 30) and the densitydependent nature of AHL signaling, we targeted benthic eukaryotes as likely sources of AHL-producing bacteria. Marine sponges in particular are notable for their diverse microbial biota (15,16,32), with bacterial densities in some sponges exceeding those in surrounding seawater by 2 to 3 orders of magnitude (7). Here, we demonstrate for the first time the production of AHLs by bacteria isolated from sponges and the apparent in situ production of AHLs within sponge tissue.Marine organisms, or portions thereof, were collected by scuba diving at depths of 1 to 10 m from several coastal locations near Sydney, in southeastern Australia. Duplicate individuals were sampled for species from the following taxa: sponges (31 species); macroalgae (30 species); and sea grasses, bryozoans, ascidians, and corals (2 species each). Direct screening of small tissue samples by the Chromobacterium violaceum CV026 bioassay (21) revealed the presence of AHLproducing bacteria in all host phyla examined (Table 1). While AHLs appeared to be widespread in marine eukaryote-bacterium associations (especially those involving sponges), there was also substantial variability (both over time and within species) in their occurrence. For example, in a separate short-term sampling program, AHLs were detected in only two out of eight sponge species tested on day 1 but were foun...