Nazia Mojib

Occurrence and distribution of capB in Antarctic microorganisms and study of its structure and regulation in the Antarctic biodegradative Pseudomonas sp. 30/3

The analysis of the cold-shock domain (CSD)-encoding genes, capB and cspA, by PCR amplification showed presence of capB in all 18 Antarctic Pseudomonas isolates, but the absence of cspA. Nucleotide sequence analysis of capB ORF from a biodegradative Pseudomonas 30/3 and its regulatory sequences including the promoter and 5′-UTR was determined and compared with the other CSD-encoding genes. Expression analysis using translational gene fusion of the putative capB promoter and its flanking sequence from Pseudomonas sp. 30/3 with lacZ′ exhibited a significant increase in β-galactosidase activity at 15 and 6°C. Unlike the expression of E. coli CspA, Pseudomonas sp. 30/3 showed a slow but steady increase of the CapB expression at 6°C. Subcellular localization of CapB at 6°C showed accumulation in and around the nucleoid whereas at 22 or 30°C, it was identified around the nucleoid as well as in the cytosol. Our study attempts to elucidate the detailed structure of capB from Pseudomonas 30/3 and the role of 5′UTR in the transcriptional regulation along with the possible role of CapB in transcription and translation suited for the cold adaptation of this bacterium in Antarctic environment.

Antimicrobial activity of PVP from an Antarctic bacterium, Janthinobacterium sp. Ant5-2, on multi-drug and methicillin resistant Staphylococcus aureus

Multiple drug resistant (MDR) and methicillin-resistant Staphylococcus aureus (MRSA) have become increasingly prevalent as a community acquired infection. As a result limited treatment options are available with conventional synthetic antibiotics. Bioprospecting natural products with potent antimicrobial activity show promise for developing new drugs against this pathogen. In this study, we have investigated the antimicrobial activity of a purple violet pigment (PVP) from an Antarctic bacterium, Janthinobacterium sp. Ant5-2 on 15 clinical MDR and MRSA strains. The colorimetric resazurin assay was employed to determine the minimum inhibitory concentration (MIC90) of PVP against MDR and MRSA. The MIC90 ranged between 1.57 µg/mL and 3.13 µg/mL, which are significantly lower than many antimicrobials tested from natural sources against this pathogen. The spectrophotometrically determined growth analysis and total microscopic counts using Live/dead® BacLight™ fluorescent stain exhibited a steady decrease in viability of both MDR and MRSA cultures following treatment with PVP at the MIC levels. In silico predictive molecular docking study revealed that PVP could be a DNA-targeting minor groove binding antimicrobial compound. The continued development of novel antimicrobials derived from natural sources with the combination of a suite of conventional antibiotics could stem the rising pandemic of MDR and MRSA along with other deadly microbial pathogens.

The antiproliferative function of violacein-like purple violet pigment (PVP) from an Antarctic Janthinobacterium sp. Ant5-2 in UV-induced 2237 fibrosarcoma.

Background  In this study, we have investigated the chemotherapeutic potential of a purple violet pigment (PVP), which was isolated from a previously undescribed Antarctic Janthinobacterium sp. (Ant5‐2), against murine UV‐induced 2237 fibrosarcoma and B16F10 melanoma cells.

Bacterial gene expression at low temperatures

Under suboptimal environmental conditions such as low temperatures, many bacteria have an extended lag phase, altered cell structures, and composition such as a less fluid (more rigid) and leaky cytoplasmic membrane. As a result, cells may die, enter into a starvation mode of metabolism or a physiologically viable but non-culturable (VBNC) state. In the latter state, the amount of gene expression per cell is virtually undetectable. In this article, gene expression under (suboptimal) low temperature conditions in non-psychrophilic environmental bacteria is examined. The pros and cons of some of the molecular methodologies for gene expression analysis are also discussed.

Structure and function of a cold shock domain fold protein, CspD, in Janthinobacterium sp. Ant5-2 from East Antarctica

A cold shock domain (CSD)-containing protein, CspD, of molecular mass ~7.28 kDa in a psychrotolerant Antarctic Janthinobacterium sp. Ant5-2 (ATCC BAA-2154) exhibited constitutive expression at 37, 22, 15, 4 and -1°C. The cspD gene encoding the CspD protein of Ant5-2 was cloned, sequenced and analyzed. The deduced protein sequence was highly similar to the conserved domains of the cold shock proteins (Csps) from bacteria belonging to the class Betaproteobacteria. Its expression was both time- and growth phase-dependent and increased when exposed to 37°C and UV radiation (UVC, dose: 1.8 and 2.8 mJ cm(-2)). The results from the electrophoretic mobility shift and subcellular localization study confirmed its single-stranded DNA-binding property. In silico analysis of the deduced tertiary structure of CspD from Ant5-2 showed a highly stable domain-swapped dimer, forming two similar monomeric Csp folds. This study established an overall framework of the structure, function and phylogenetic analysis of CspD from an Antarctic Janthinobacterium sp. Ant5-2, which may facilitate and stimulate the study of CSD fold proteins in the class Betaproteobacteria.

Microbial Communities and Their Predicted Metabolic Functions in Growth Laminae of a Unique Large Conical Mat from Lake Untersee, East Antarctica

In this study, we report the distribution of microbial taxa and their predicted metabolic functions observed in the top (U1), middle (U2), and inner (U3) decadal growth laminae of a unique large conical microbial mat from perennially ice-covered Lake Untersee of East Antarctica, using NextGen sequencing of the 16S rRNA gene and bioinformatics tools. The results showed that the U1 lamina was dominated by cyanobacteria, specifically Phormidium sp., Leptolyngbya sp., and Pseudanabaena sp. The U2 and U3 laminae had high abundances of Actinobacteria, Verrucomicrobia, Proteobacteria, and Bacteroidetes. Closely related taxa within each abundant bacterial taxon found in each lamina were further differentiated at the highest taxonomic resolution using the oligotyping method. PICRUSt analysis, which determines predicted KEGG functional categories from the gene contents and abundances among microbial communities, revealed a high number of sequences belonging to carbon fixation, energy metabolism, cyanophycin, chlorophyll, and photosynthesis proteins in the U1 lamina. The functional predictions of the microbial communities in U2 and U3 represented signal transduction, membrane transport, zinc transport and amino acid-, carbohydrate-, and arsenic- metabolisms. The Nearest Sequenced Taxon Index (NSTI) values processed through PICRUSt were 0.10, 0.13, and 0.11 for U1, U2, and U3 laminae, respectively. These values indicated a close correspondence with the reference microbial genome database, implying high confidence in the predicted metabolic functions of the microbial communities in each lamina. The distribution of microbial taxa observed in each lamina and their predicted metabolic functions provides additional insight into the complex microbial ecosystem at Lake Untersee, and lays the foundation for studies that will enhance our understanding of the mechanisms responsible for the formation of these unique mat structures and their evolutionary significance.

Diversity of bacterial communities in the lakes of Schirmacher Oasis, Antarctica

Extreme conditions such as low temperature, aridness, low availability of organic matter, high salinity and UV-radiation in terrestrial Antarctica are key factors limiting the habitation of biotic communities and ecosystem dynamics. In recent studies, it has been discovered that the bacterial communities are highly diverse and distributed widely in the extreme ecosystem of Antarctica. Besides available morphometric data, geology, and thermal profile, limited study on the microbial identification, phylogenetic analysis, diversity and distribution of microorganisms in different lakes of Schirmacher Oasis in East Antarctica has been reported. The objective of this study was to assess the microbial biodiversity and distribution using culture-independent and culture-dependent methodologies based upon bacterial 16S rRNA gene analysis in three categories of lakes, i.e., the land-locked (L), epi-shelf (E), and pro-glacial (P) lakes in Schirmacher Oasis. The water and ice samples were collected during the 2008 Tawani International Scientific Expedition. Direct culturing of the samples on R2A agar media exhibited a wide variety of pigmented bacteria. Two of the pigmented bacteria that were cultured belong to the genera, Hymenobacter, and Flavobacterium. Cultureindependent methodology of one of the land-locked lakes L27C identified a rich microbial diversity consisting of six different phyla of bacteria. The majority of bacteria (56%) belong to the Class γ-proteobacteria within the phylum Proteobacteria. Within the Class γ-proteobacteria, Acinetobacter dominated (48%) the total microbial load. Characterization of the microbial diversity within the three different types of Antarctic lakes is important because it will help give us a better understanding of the survival mechanisms and the functionality of these bacteria in extremely cold and harsh Antarctic ecosystems.

Diversity and cold adaptation of microorganisms isolated from the Schirmacher Oasis, Antarctica

We have investigated the feasibility of the PCR amplification of the 16S rRNA genes from eubacteria and Archea on samples collected on Whatman FTA filters from Schirmacher Oasis for the study of culture-independent analysis of the microbial diversity. Both conventional PCR and real-time TaqmaTM PCR successfully amplified the targeted genes. A number of diverse groups of psychrotolerant microorganisms with various pigments have been isolated when cultured on agar medium. 16S rRNA gene analysis of these isolates helped us to identify closest taxonomic genus Pseudomonas, Frigoribacterium, Arthrobacter, Flavobacterium, and Janthinobacterium. It is possible that the pigments play protective role from solar UV radiation, which is prevalent in Antarctic continent especially during Austral summer months. Study of the expression of cold adaptive protein CapB and ice-binding protein IBP using western blots showed positive detection of both or either of these proteins in 6 out of 8 isolates. Since the CapB and IBP protein structure greatly varies in microorganisms, it is possible that the 2 isolates with negative results could have a different class of these proteins. The expression of the CapB and the IBP in these isolates suggest that these proteins are essential for the survival in the Antarctic cold and subzero temperatures and protect themselves from freeze-damage. The current study provided sufficient data to further investigate the rich and diverse biota of psychrotolerant extremophiles in the Antarctic Schirmacher Oasis using both culture-independent and culture-based approaches; and understand the mechanisms of cold tolerance.