Fifty-seven proteobacterium species were successfully isolated from soils of Barrientos Island of the Antarctic using 11 different isolation media. Analysis of 16S rDNA sequencing of these isolates showed that they belonged to eight different genera, namely Bradyrhizobium, Sphingomonas, Methylobacterium, Caulobacter, Paracoccus, Ralstonia, Rhizobium, and Staphylococcus. All isolates were studied for capability of producing antimicrobial and antifungal secondary metabolites using high-throughput screening models. Approximately 23 (13/57) and 2% (1/57) of isolates inhibited growth of Candida albicans ATCC 10231(T) and Staphylococcus aureus ATCC 51650(T), respectively. These results indicated that proteobacterium species isolates from Antarctic could serve as potential source of useful bioactive metabolites. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting produced nine clusters and 13 single isolates, with a high D value of 0.9248. RAPD fingerprinting produced six clusters and 13 single isolates, with a relatively low D value of 0.7776. ERIC-PCR analysis proved to have better discrimination capability than RAPD analysis and generated better clustering for all proteobacterium species isolates. We conclude that ERIC-PCR is a robust, reliable and rapid molecular typing method for discriminating different genera of proteobacteria.
Proteobacteria are known to communicate via signaling molecules and this process is known as quorum sensing. The most commonly studied quorum sensing molecules are N-acylhomoserine lactones (AHLs) that consists of a homoserine lactone moiety and an N-acyl side chain with various chain lengths and degrees of saturation at the C-3 position. We have isolated a bacterium, RB-44, from a site which was formally a landfill dumping ground. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis, this isolate was identified as a Pandoraea sp.which was then screened for AHL production using biosensors which indicated its quorum sensing properties. To identify the AHL profile of Pandoraea sp. RB-44, we used high resolution tandem mass spectrometry confirming that this isolate produced N-octanoylhomoserine lactone (C8-HSL). To the best of our knowledge, this is the first report that showed quorum sensing activity exhibited by Pandoraea sp. Our data add Pandoraea sp. to the growing number of bacteria that possess QS systems.
Geographical variation in soil bacterial community structure in 26 tropical forests in Southeast Asia (Malaysia, Indonesia and Singapore) and two temperate forests in Japan was investigated to elucidate the environmental factors and mechanisms that influence biogeography of soil bacterial diversity and composition. Despite substantial environmental differences, bacterial phyla were represented in similar proportions, with Acidobacteria and Proteobacteria the dominant phyla in all forests except one mangrove forest in Sarawak, although highly significant heterogeneity in frequency of individual phyla was detected among forests. In contrast, species diversity (α-diversity) differed to a much greater extent, being nearly six-fold higher in the mangrove forest (Chao1 index = 6,862) than in forests in Singapore and Sarawak (~1,250). In addition, natural mixed dipterocarp forests had lower species diversity than acacia and oil palm plantations, indicating that aboveground tree composition does not influence soil bacterial diversity. Shannon and Chao1 indices were correlated positively, implying that skewed operational taxonomic unit (OTU) distribution was associated with the abundance of overall and rare (singleton) OTUs. No OTUs were represented in all 28 forests, and forest-specific OTUs accounted for over 70% of all detected OTUs. Forests that were geographically adjacent and/or of the same forest type had similar bacterial species composition, and a positive correlation was detected between species divergence (β-diversity) and direct distance between forests. Both α- and β-diversities were correlated with soil pH. These results suggest that soil bacterial communities in different forests evolve largely independently of each other and that soil bacterial communities adapt to their local environment, modulated by bacterial dispersal (distance effect) and forest type. Therefore, we conclude that the biogeography of soil bacteria communities described here is non-random, reflecting the influences of contemporary environmental factors and evolutionary history.
Soil bacterial community structures of six dominant phyla (Acidobacteria, Proteobacteria, Verrucomicrobia, Planctomycetes, Bacteroidetes and Actinobacteria) and unclassified bacteria detected in tropical Sarawakian and temperate Japanese forests were compared based on 16S rRNA gene sequence variation. The class composition in each phylum was similar among the studied forests; however, significant heterogeneities of class frequencies were detected. Acidobacteria and Proteobacteria were the most dominant phyla in all six forests, but differed in the level of bacterial species diversity, pattern of species occurrence and association pattern of species composition with physicochemical properties in soil. Species diversity among Acidobacteria was approximately half that among Proteobacteria, based on the number of clusters and the Chao1 index, even though a similar number of sequence reads were obtained for these two phyla. In contrast, species diversity within Planctomycetes and Bacteroidetes was nearly as high as within Acidobacteria, despite many fewer sequence reads. The density of species (the number of sequence reads per cluster) correlated negatively with species diversity, and species density within Acidobacteria was approximately twice that within Proteobacteria. Although the percentage of forest-specific species was high for all bacterial groups, sampling site-specific species varied among bacterial groups, indicating limited inter-forest migration and differential movement of bacteria in forest soil. For five of the seven bacterial groups, including Acidobacteria, soil pH appeared to strongly influence species composition, but this association was not observed for Proteobacterial species. Topology of UPGMA trees and pattern of NMDS plots among the forests differed among the bacterial groups, suggesting that each bacterial group has adapted and evolved independently in each forest.
A novel chemically defined medium, named KG medium, supplemented with N-3-oxo-hexanoylhomoserine lactone (3-oxo-C6-HSL), an acylhomoserine lactone (AHL) used as signalling molecules in Gram-negative bacterial cell-to-cell communication, as the sole source of carbon and nitrogen, was designed and successfully used for the enrichment and isolation of AHL-degrading bacteria. A 3-oxo-C6-HSL-degrading bacterium, 13sw7, was isolated from sewage after six enrichment transfers in the 3-oxo-C6-HSL-containing KG medium. On the basis of the almost complete 16S ribosomal DNA sequence, isolate 13sw7 was clustered with unculturable beta-proteobacteria. This study indicates that the AHL-containing KG medium is effective in isolating AHL-degrading bacteria, including those previously considered unculturable, from environmental sources. To the best of our knowledge, this is the first documentation of the isolation of an AHL-degrading proteobacterium from sewage.
Banana is one of the most important fruits cultivated in Malaysia, and it provides many health benefits. However, bacterial wilt disease, which attacks bananas, inflicts major losses on the banana industry in Malaysia. To understand the complex interactions of the microbiota of bacterial wilt-diseased banana plants, we first determined the bacterial communities residing in the pseudostems of infected (symptomatic) and diseased-free (non-symptomatic) banana plants. We characterized the associated microorganisms using the targeted 16S rRNA metagenomics sequencing on the Illumina MiSeq platform. Taxonomic classifications revealed 17 and nine known bacterial phyla in the tissues of non-symptomatic and symptomatic plants, respectively. Cyanobacteria and Proteobacteria (accounted for more than 99% of the 16S rRNA gene fragments) were the two most abundant phyla in both plants. The five major genera found in both plant samples were Ralstonia, Sphingomonas, Methylobacterium, Flavobacterium, and Pseudomonas. Ralstonia was more abundant in symptomatic plant (59% out of the entire genera) as compared to those in the non-symptomatic plant (only 36%). Our data revealed that 102 bacterial genera were only assigned to the non-symptomatic plant. Overall, this study indicated that more diverse and abundant microbiota were associated with the non-symptomatic bacterial wilt-diseased banana plant as compared to the symptomatic plant. The higher diversity of endophytic microbiota in the non-symptomatic banana plant could be an indication of pathogen suppression which delayed or prevented the disease expression. This comparative study of the microbiota in the two plant conditions might provide caveats for potential biological control strategies.