Abhrajyoti Ghosh

Culture independent molecular analysis of bacterial communities in the mangrove sediment of Sundarban, India

BackgroundSundarban is the worlds largest coastal sediment comprising of mangrove forest which covers about one million hectares in the south-eastern parts of India and southern parts of Bangladesh. The microbial diversity in this sediment is largely unknown till date. In the present study an attempt has been made to understand the microbial diversity in this sediment using a cultivation-independent molecular approach.ResultsTwo 16 S rRNA gene libraries were constructed and partial sequencing of the selected clones was carried out to identify bacterial strains present in the sediment. Phylogenetic analysis of partially sequenced 16 S rRNA gene sequences revealed the diversity of bacterial strains in the Sundarban sediment. At least 8 different bacterial phyla were detected. The major divisions of detected bacterial phyla were Proteobacteria (alpha, beta, gamma, and delta), Flexibacteria (CFB group), Actinobacteria, Acidobacteria, Chloroflexi, Firmicutes, Planctomycetes and Gammatimonadates.ConclusionThe gammaproteobacteria were found to be the most abundant bacterial group in Sundarban sediment. Many clones showed similarity with previously reported bacterial lineages recovered from various marine sediments. The present study indicates a probable hydrocarbon and oil contamination in this sediment. In the present study, a number of clones were identified that have shown similarity with bacterial clones or isolates responsible for the maintenance of the S-cycle in the saline environment.

Molecular analysis of the crenarchaeal flagellum

The ability to move towards favourable conditions provides fundamental advantages to organisms. Interestingly, flagella as motility structures evolved independently in the bacterial and the archaeal kingdom. Whereas bacterial flagella have been intensively studied, our knowledge regarding the archaeal counterpart is mostly restricted to Euryarchaeota rather than crenarchaeal flagella. We therefore investigated the flagellar assembly system of the crenarchaeal model organism Sulfolobus acidocaldarius in vivo. Promoter studies and qRT‐PCR analyses of the flagella gene cluster provided evidence that the expression of the fla genes was induced by tryptone starvation. Moreover, we confirmed presence of a secondary fla promoter within the flaB gene that regulates the transcription of downstream genes flaX‐J. Markerless in‐frame deletions for all fla genes encoded in the fla gene cluster were constructed. Western blot analysis of all fla deletion strains suggested hierarchical protein interactions during the archaeal flagella assembly. Moreover, functional analysis by thermomicroscopy revealed non‐motile cells for each of the mutant strains. Electron micrographs demonstrated that lack of motility coincided with the loss of flagellar assembly. Thus we demonstrated that all seven fla genes are essential for crenarchaeal flagellum assembly and function.

Archaeal type IV pilus-like structures--evolutionarily conserved prokaryotic surface organelles.

In both bacteria and Archaea, the biosynthesis of type IV pilus-related structures involves a set of core components, including a prepilin peptidase that specifically processes precursors of pilin-like proteins. Although in silico analyses showed that most sequenced archaeal genomes encode predicted pilins and conserved pilus biosynthesis components, recent in vivo analyses of archaeal pili in genetically tractable crenarchaea and euryarchaea revealed Archaea-specific type IV pilus functions and biosynthesis components. Studies in a variety of archaeal species will reveal which type IV pilus-like structures are common in Archaea and which are limited to certain species within this domain. The insights gleaned from these studies may also elucidate the roles played by these types of structures in adapting to specific environments.

Influence of cell surface structures on crenarchaeal biofilm formation using a thermostable green fluorescent protein.

The thermoacidophilic crenarchaeote Sulfolobus acidocaldarius displays three distinct type IV pili-like structures on its surface: (i) the flagellum, (ii) the UV-induced pili and (iii) the adhesive pili. In bacteria, surface appendages play an important role in the spatial organization of cells from initial surface attachment to the development of mature community structures. To investigate the influence of the diverse set of type IV pili-like structures in S. acidocaldarius, single, double and triple mutants lacking the cell surface appendages were constructed and analysed for their behaviour in attachment assays and during biofilm formation. A heat stable green fluorescent protein was employed the first time in a hyperthermophilic archaeon. A codon adjusted eCGP123 was expressed to study mixed biofilms of different deletion mutants to understand the interplay of the surface structures during biofilm formation. During this process the deletion of the adhesive pili and UV-induced pili led to the most pronounced effects, either an increase in cell density or increased cluster formation respectively. However, all three cell surface appendages played a role in the colonization of surfaces and only the interplay of all three appendages leads to the observed wild-type biofilm phenotype.

Bacterial diversity of east Calcutta wet land area: possible identification of potential bacterial population for different biotechnological uses

The extent of microbial diversity in nature is still largely unknown, suggesting that there might be many more useful products yet to be identified from soil microorganisms. This insight provides the scientific foundation for a renewed interest in examining soil microorganisms for novel commercially important products. This has led us to access the metabolic potential of soil microorganisms via cultivation strategy. Keeping this in mind, we have performed a culture-dependent survey of important soil bacterial community diversity in East Calcutta Wetland area (Dhapa Landfill Area). We describe isolation of 38 strains, their phenotypic and biochemical characterization, and finally molecular identification by direct sequencing of polymerase chain reaction (PCR)-amplified 16S rRNA gene products. We have isolated and identified strains able to fix nitrogen, produce extracellular enzymes like protease, cellulase, xylanase, and amylase, and solubilize inorganic phosphates. Some isolates can synthesize extracellular insecticidal toxins. We find a good correlation between biochemical and phenotypic behavior and the molecular study using 16S rRNA gene of the isolates. Furthermore, our findings clearly indicate the composition of cultivable soil bacteria in East Calcutta Wetland Area.

Structure and function of the adhesive type IV pilus of Sulfolobus acidocaldarius

Archaea display a variety of type IV pili on their surface and employ them in different physiological functions. In the crenarchaeon Sulfolobus acidocaldarius the most abundant surface structure is the aap pilus (archaeal adhesive pilus). The construction of in frame deletions of the aap genes revealed that all the five genes (aapA, aapX, aapE, aapF, aapB) are indispensible for assembly of the pilus and an impact on surface motility and biofilm formation was observed. Our analyses revealed that there exists a regulatory cross-talk between the expression of aap genes and archaella (formerly archaeal flagella) genes during different growth phases. The structure of the aap pilus is entirely different from the known bacterial type IV pili as well as other archaeal type IV pili. An aap pilus displayed 3 stranded helices where there is a rotation per subunit of ∼138° and a rise per subunit of ∼5.7 Å. The filaments have a diameter of ∼110 Å and the resolution was judged to be ∼9 Å. We concluded that small changes in sequence might be amplified by large changes in higher-order packing. Our finding of an extraordinary stability of aap pili possibly represents an adaptation to harsh environments that S. acidocaldarius encounters.

Diversity, assembly and regulation of archaeal type IV pili-like and non-type-IV pili-like surface structures.

Archaea have evolved fascinating surface structures allowing rapid adaptation to changing environments. The archaeal surface appendages display such diverse biological roles as motility, adhesion, biofilm formation, exchange of genetic material and species-specific interactions and, in turn, increase fitness of the cells. Intriguingly, despite sharing the same functions with their bacterial counterparts, the assembly mechanism of many archaeal surface structures is rather related to assembly of bacterial type IV pili. This review summarizes our state-of-the-art knowledge about unique structural and biochemical properties of archaeal surface appendages with a particular focus on archaeal type IV pili-like structures. The latter comprise not only widely distributed archaella (formerly known as archaeal flagella), but also different highly specialized archaeal pili, which are often restricted to certain species. Recent findings regarding assembly mechanisms, structural aspects and physiological roles of these type IV pili-like structures will be discussed in detail. Recently, first regulatory proteins involved in transition from both planktonic to sessile lifestyle and in assembly of archaella were identified. To conclude, we provide novel insights into regulatory mechanisms underlying the assembly of archaeal surface structures.

Thermodynamic characterization of a highly thermoactive extracellular pectate lyase from a new isolate Bacillus pumilus DKS1

An extracellular pectate lyase (EC 4.2.2.2) was purified from the culture filtrate of a newly isolated Bacillus pumilus DKS1 grown in pectin containing medium. Using ion-exchange and gel filtration chromatography, this enzyme was purified and found to have a molecular weight of around 35kDa. The purified enzyme exhibited maximal activity at a temperature of 75 degrees C and pH 8.5. The presence of 1mM calcium and manganese enhanced pectate lyase activity and was strongly inhibited by zinc, nickel and EDTA. The thermal inactivation studies revealed an entropy-enthalpy compensation pattern below a critical temperature. The alkaliphilicity and high thermostability of this pectate lyase may have potential implications in fibre degumming.

FlaX, A Unique Component of the Crenarchaeal Archaellum, Forms Oligomeric Ring-shaped Structures and Interacts with the Motor ATPase FlaI

Background: FlaX is essential for archaella assembly in Sulfolobus acidocaldarius and its stability is dependent on FlaI-J. Results: FlaX assembles into ring like oligomers and interacts with the motor ATPase FlaI. Conclusion: FlaX oligomers depend on the presence of its C-terminal domain, which also interacts with FlaI. Significance: The first structural analysis of an archaellum subunit, a rotating type IV pilus structure. Archaella are the archaeal motility structure, which are structurally similar to Gram-negative bacterial type IV pili but functionally resemble bacterial flagella. Structural and biochemical data of archaellum subunits are missing. FlaX, a conserved subunit in crenarchaeal archaella, formed high molecular weight complexes that adapted a ring-like structure with an approximate diameter of 30 nm. The C terminus of FlaX was not only involved in the oligomerization, but also essential for FlaX interaction with FlaI, the bifunctional ATPase that is involved in assembly and rotation of the archaellum. This study gives first insights in the assembly apparatus of archaella.

Complementation of Sulfolobus solfataricus PBL2025 with an α-mannosidase: effects on surface attachment and biofilm formation

Compared to Sulfolobus solfataricus P2, the S. solfataricus mutant PBL2025 misses 50 genes (SSO3004-3050), including genes coding for a multitude of enzymes possibly involved in sugar degradation or metabolism. We complemented PBL2025 with two of the missing proteins, the α-mannosidase (SSO3006, Ssα-man) and the β-galactosidase LacS (SSO3019), and performed comparative fluorescence microscopy and confocal laser scanning microscopy to analyze the recombinant strains. We demonstrated that the Ssα-man complemented strain resembled the S. solfataricus P2 behavior with respect to attachment of cells to glass and growth of cells in static biofilms. During expression of the Ssα-man, but not LacS, glucose and mannose-containing extracellular polymeric substance (EPS) levels changed in the recombinant strain during surface attachment and biofilm formation. These results suggest that the Ssα-man might be involved in the modulation of the EPS composition and/or in the de-mannosylation of the glycan tree, which is attached to extracellular glycosylated proteins in S. solfataricus. On the other hand, LacS expression in PBL2025 reduced the carbohydrate content of the isolated total EPS implying a role in the modulation of the produced EPS during static biofilm formation. These are the first enzymes identified as playing a role in archaeal EPS formation.