Bacteria Cultivated From Sponges and Bacteria Not Yet Cultivated From Sponges—A Review

The application of high-throughput microbial community profiling as well as “omics” approaches unveiled high diversity and host-specificity of bacteria associated with marine sponges, which are renowned for their wide range of bioactive natural products. However, exploration and exploitation of bioactive compounds from sponge-associated bacteria have been limited because the majority of the bacteria remains recalcitrant to cultivation. In this review, we (i) discuss recent/novel cultivation techniques that have been used to isolate sponge-associated bacteria, (ii) provide an overview of bacteria isolated from sponges until 2017 and the associated culture conditions and identify the bacteria not yet cultured from sponges, and (iii) outline promising cultivation strategies for cultivating the uncultivated majority of bacteria from sponges in the future. Despite intensive cultivation attempts, the diversity of bacteria obtained through cultivation remains much lower than that seen through cultivation-independent methods, which is particularly noticeable for those taxa that were previously marked as “sponge-specific” and “sponge-enriched.” This poses an urgent need for more efficient cultivation methods. Refining cultivation media and conditions based on information obtained from metagenomic datasets and cultivation under simulated natural conditions are the most promising strategies to isolate the most wanted sponge-associated bacteria.

Screening of Neutrophil Activating Factors from a Metagenome Library of Sponge-Associated Bacteria

Marine sponge-associated bacteria are known as bio-active compound produce. We have constructed metagenome libraries of the bacteria and developed a metagenomic screening approach. Activity-based screening successfully identified novel genes and novel enzymes; however, the efficiency was only in 1 out of 104 clones. Therefore, in this study, we thought that bioinformatics could help to reduce screening efforts, and combined activity-based screening with database search. Neutrophils play an important role for the immune system to recognize excreted bacterial by-products as chemotactic factors and are recruited to infection sites to kill pathogens via phagocytosis. These excreted by-products are considered critical triggers that engage the immune system to mount a defense against infection, and identifying these factors may guide developments in medicine and diagnostics. We focused on genes encoding amino acid ligase and peptide synthetase and selected from an in-house sponge metagenome database. Cell-free culture medium of each was used in a neutrophil chemiluminescence assay in luminol reaction. The clone showing maximum activity had a genomic sequence expected to produce a molecule like a phospho-N-acetylmuramyl pentapeptide by the metagenome fragment analysis.

Isolation and Screening the Symbiont Bacteria of the Sponge Dragmacidon sp from Manado Bay, North Sulawesi that Producing Chitinase and Protease

Enzymes are important in the technology industry and hydrolytic enzymes, such as chitinase and protease are commonly used for it. Various types of microorganisms such as bacteria can produce hydrolytic enzymes. Sponge-associated bacteria are excellent sources of extracellular hydrolytic enzymes because the surface and internal spaces of sponges are richer in nutrients. The aim of this study was to isolate and screen the bacteria of the sponge Dragmacidon sp symbiotic from Manado Bay, North Sulawesi that producing chitinase and protease Symbiont bacteria were grown in Zobell 1226 E medium with a dilution of 10-4. Bacterial isolation was carried out based on the morphological characteristics of the colony. Chitinase and protease activity was carried out by growing each bacterial isolate in chitin and protein media at 36oC for 48 hours. Chitinase and protease activities were indicated by the formation of a clear zone around the bacterial colony, however, the clear zone for chitinase activity was observed after pouring the Lugol's solution. Based on this study, 8 isolates bacteria of the symbiotic spongy Dragmacidon sp from Manado Bay, North Sulawesi were isolated based on morphological characteristics. The colony of the bacteria is generally white with an irregular shape. Four isolates, namely 1, 2, 3, and 8 had chitinase activity with chitinolytic indexes were 1.7; 1.5; 1.4, and 1.3, respectively. Six isolates, namely 1, 2, 3, 4, 5, and 6 had protease activity with proteolytic indexes were 1.4; 1.8; 3.1; 1.3; 1.8; and 2.5, respectively.Keywords: Bacteria; Chitinolytic; Proteolytic; Symbiont; SpongeAbstrakEnzim menempati posisi penting dalam bidang teknologi dan industri. Enzim yang banyak digunakan dalam bidang industri adalah enzim hidrolase. Enzim dapat diisolasi dari berbagai jenis mikroorganisme seperti bakteri. Bakteri yang berasosiasi dengan spons merupakan sumber enzim hidrolitik ekstraseluler yang sangat baik karena permukaan dan ruang internal spons lebih kaya nutrisi. Penelitian ini bertujuan untuk mengisolasi dan menguji aktivitas kitinase dan protease bakteri simbion spons Dragmacidon sp dari Teluk Manado. Bakteri simbion spons ditumbuhkan dalam media Zobell 1226 E pada pengenceran 10-4. Isolasi bakteri dilaksanakan berdasakan karakteristik morfologi. Aktivitas kitinase dan protease dilaksanakan dengan menumbuhkan setiap isolat bakteri dalam media kitin dan protein pada suhu 36oC selama 48 jam. Aktivitas kitinase dan protease ditandai dengan terbentuknya zona bening di sekitar koloni bakteri yang mana untuk kitinase diamati setelah diberi larutan lugol. Berdasarkan penelitian ini, 8 isolat bakeri simbion spon Dragmacidon sp dari Teluk Manado, Sulawesi Utara berhasil diisolasi berdasarkan karakteristik morfologi. Isolat bakteri umumnya berwarna putih dengan bentuk ireguller. Empat isolat yakni 1, 2, 3, dan 8 memiliki aktivitas kitinase dan enam isolat yakni 1, 2, 3, 4, 5, dan 6 yang memiliki aktivitas protease. Indeks kitinolitik dari masing-masing keempat isolat bakteri secara berturut turut adalah 1,7; 1,5; 1,4; dan 1,3 dengan kategori bernilai rendah dan indeks proteolitik adalah 1,4; 1,8; 3,1; 1,3; 1,8; dan 2,5 dengan kategori bernilai rendah sampai tinggi.Kata kunci: Bakteri; Kitinolitik; Proteolitik; Simbion; Spons

Diversity and Antimicrobial Activity of Vietnamese Sponge-Associated Bacteria

This study aimed to assess the diversity and antimicrobial activity of cultivable bacteria associated with Vietnamese sponges. In total, 460 bacterial isolates were obtained from 18 marine sponges. Of these, 58.3% belonged to Proteobacteria, 16.5% to Actinobacteria, 18.0% to Firmicutes, and 7.2% to Bacteroidetes. At the genus level, isolated strains belonged to 55 genera, of which several genera, such as Bacillus, Pseudovibrio, Ruegeria, Vibrio, and Streptomyces, were the most predominant. Culture media influenced the cultivable bacterial composition, whereas, from different sponge species, similar cultivable bacteria were recovered. Interestingly, there was little overlap of bacterial composition associated with sponges when the taxa isolated were compared to cultivation-independent data. Subsequent antimicrobial assays showed that 90 isolated strains exhibited antimicrobial activity against at least one of seven indicator microorganisms. From the culture broth of the isolated strain with the strongest activity (Bacillus sp. M1_CRV_171), four secondary metabolites were isolated and identified, including cyclo(L-Pro-L-Tyr) (1), macrolactin A (2), macrolactin H (3), and 15,17-epoxy-16-hydroxy macrolactin A (4). Of these, compounds 2-4 exhibited antimicrobial activity against a broad spectrum of reference microorganisms.

Bacteria Isolated From the Antarctic Sponge Iophon sp. Reveals Mechanisms of Symbiosis in Sporosarcina, Cellulophaga, and Nesterenkonia

Antarctic sponges harbor a diverse range of microorganisms that perform unique metabolic functions for nutrient cycles. Understanding how microorganisms establish functional sponge–microbe interactions in the Antarctic marine ecosystem provides clues about the success of these ancient animals in this realm. Here, we use a culture-dependent approach and genome sequencing to investigate the molecular determinants that promote a dual lifestyle in three bacterial genera Sporosarcina, Cellulophaga, and Nesterenkonia. Phylogenomic analyses showed that four sponge-associated isolates represent putative novel bacterial species within the Sporosarcina and Nesterenkonia genera and that the fifth bacterial isolate corresponds to Cellulophaga algicola. We inferred that isolated sponge-associated bacteria inhabit similarly marine sponges and also seawater. Comparative genomics revealed that these sponge-associated bacteria are enriched in symbiotic lifestyle-related genes. Specific adaptations related to the cold Antarctic environment are features of the bacterial strains isolated here. Furthermore, we showed evidence that the vitamin B5 synthesis-related gene, panE from Nesterenkonia E16_7 and E16_10, was laterally transferred within Actinobacteria members. Together, these findings indicate that the genomes of sponge-associated strains differ from other related genomes based on mechanisms that may contribute to the life in association with sponges and the extreme conditions of the Antarctic environment.

A Metataxonomic Approach Reveals Diversified Bacterial Communities in Antarctic Sponges

Marine sponges commonly host a repertoire of bacterial-associated organisms, which significantly contribute to their health and survival by producing several anti-predatory molecules. Many of these compounds are produced by sponge-associated bacteria and represent an incredible source of novel bioactive metabolites with biotechnological relevance. Although most investigations are focused on tropical and temperate species, to date, few studies have described the composition of microbiota hosted by Antarctic sponges and the secondary metabolites that they produce. The investigation was conducted on four sponges collected from two different sites in the framework of the XXXIV Italian National Antarctic Research Program (PNRA) in November–December 2018. Collected species were characterized as Mycale (Oxymycale) acerata, Haliclona (Rhizoniera) dancoi, Hemigellius pilosus and Microxina sarai by morphological analysis of spicules and amplification of four molecular markers. Metataxonomic analysis of these four Antarctic sponges revealed a considerable abundance of Amplicon Sequence Variants (ASVs) belonging to the phyla Proteobacteria, Bacteroidetes, Actinobacteria and Verrucomicrobia. In particular, M. (Oxymycale) acerata, displayed several genera of great interest, such as Endozoicomonas, Rubritalea, Ulvibacter, Fulvivirga and Colwellia. On the other hand, the sponges H. pilosus and H. (Rhizoniera) dancoi hosted bacteria belonging to the genera Pseudhongella, Roseobacter and Bdellovibrio, whereas M. sarai was the sole species showing some strains affiliated to the genus Polaribacter. Considering that most of the bacteria identified in the present study are known to produce valuable secondary metabolites, the four Antarctic sponges could be proposed as potential tools for the discovery of novel pharmacologically active compounds.