A microbiologist's odyssey: Bacterial viruses to photosynthetic bacteria

Perspective can be defined as the relationships or relative importance of facts or matters from any special point of view. Thus, my Personal perspective reflects the threads I followed in a 50-year journey of research in the complex tapestry of bioenergetics and various aspects of microbial metabolism. An early interest in biochemical and microbial evolution led to the fertile hunting grounds of anoxygenic photosynthetic bacteria. Viewed as a physiological class, these organisms show remarkable metabolic versatility in that certain individual species are capable of using all the known major types of energy conversion (photosynthetic, respiratory, and fermentative) to support growth. Since such anoxyphototrophs are readily amenable to molecular genetic/biological manipulation, it can be expected that they will eventually provide important clues for unraveling the evolutionary relationships of the several kinds of energy conversion. I gradually came to believe that understanding the evolution of phototrophs would require detailed knowledge not only of how light is converted to chemical energy, but also of a) pathways of monomer production from extracellular sources of carbon and nitrogen and b) mechanisms cells use for integrating ATP regeneration with the energy-requiring biosyntheses of biological macromolecules. Serendipic observation of photoproduction of H₂ from organic compounds by Rhodospirillum rubrum in 1949 led to discovery of N₂ fixation by anoxyphototrophs, and this capacity was later exploited for the isolation of hitherto unknown species of photosynthetic prokaryotes, including the heliobacteria. Recent studies on the reaction centers of the heliobacteria suggest the possibility that these bacteria are descendents of early phototrophs that gave rise to oxygenic photosynthetic organisms.Abbreviations AMP adenosine monophosphate - ADP adenosine diphosphate - ATP adenosine triphosphate - ATPase adenosine triphosphatase - Bchl bacteriochlorophyll - DMSO dimethyl sulfoxide - NADH reduced nicotinamide adenine dinucleotide - nif ^– genes for dinitrogen fixation - Nif^– bacterial mutants incapable of dinitrogen fixation - O/R oxidation/reduction - P_i inorganic orthophosphate - R. capsulatus Rhodobacter capsulatus - R. sphaeroides Rhodobacter sphaeroides - Rps. Rhodopseudomonas - TMAO trimethyl amine-N-oxide Written at the invitation of Govindjee.     

Mouse erythroid cells express multiple putative RNA helicase genes exhibiting high sequence conservation from yeast to mammals

RNA secondary structure is a critical determinant of RNA function in ribosome assembly, pre-mRNA splicing, mRNA translation and RNA stability. The ‘DEAD/H’ family of putative RNA helicases may help regulate these processes by utilizing intrinsic RNA-dependent ATPase activity to catalyze conformational changes in RNA secondary structure. To investigate the repertoire of DEAD/H box proteins expressed in mammals, we used PCR techniques to clone from mouse erythroleukemia (MEL) cells three new DEAD box cDNAs with high similarity to known yeast (Saccharomyces cerevisiae) genes. mDEAD2 and mDEAD3 (mouse DEAD box proteins) are >95% identical to mouse PL10 but exhibit differential tissue-specific expression patterns; mDEAD2 and mDEAD3 are also approx. 70% identical (at the aa level) to yeast DED1 and DBP1 proteins. Members of this DEAD box subclass contain C-terminal domains with high content of Arg, Ser, Gly and Phe, reminiscent of the RS domain in several Drosophila and mammalian splicing factors. mDEAD5 belongs to a second class related to translation initiation factors from yeast (TIF1/TIF2) and mammals (eIF-4A); this class contains a novel conserved peptide motif not found in other DEAD box proteins. Northern blotting shows that mDEAD5 is differentially expressed in testis vs. somatic tissues. Thus, mouse erythroid cells produce two highly conserved families of putative RNA helicases likely to play important roles in RNA metabolism and gene expression.     

Molecular analysis of genomic DNA-mediated transformation in Zea mays

Total genomic DNA isolated from maize hygromycin B resistant cell line(hygr-G204) was used to transform the maize hygromycin B sensitive cell line(hygs-G204) to the hygr-phenotype using polyethyleneglycol treatment and the transformed calli were selected using hygromycin B. The primary transformant maize plants were regenerated and analysed at the molecular level using DNA hybridization, transgenome rescue and histochemical β-glucuronidase assay. The results indicated that genomic DNA-mediated transformation can lead to transfer, expression and stable integration of a DNA fragment into the host genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of two M17 family members in Escherichia coli, Peptidase A and Peptidase B

Escherichia coli encodes two aminopeptidases belonging to the M17 family: Peptidase A (PepA) and Peptidase B (PepB). To gain insights into their substrate specificities, PepA or PepB were overexpressed in ΔpepN, which shows greatly reduced activity against the majority of amino acid substrates. Overexpression of PepA or PepB increases catalytic activity of several aminopeptidase substrates and partially rescues growth of ΔpepN during nutritional downshift and high temperature stress. Purified PepA and PepB display broad substrate specificity and Leu, Lys, Met and Gly are preferred substrates. However, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and insulin B chain peptide. Importantly, this strategy, i.e. overexpression of peptidases in ΔpepN and screening a panel of substrates for cleavage, can be used to rapidly identify peptidases with novel substrate specificities encoded in genomes of different organisms.     

Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) Is Upregulated in Breast Epithelial–Mesenchymal Transition and Responds to Oxidative Stress

Breast cancer cells that have undergone partial epithelial–mesenchymal transition (EMT) are believed to be more invasive than cells that have completed EMT. To study metabolic reprogramming in different mesenchymal states, we analyzed protein expression following EMT in the breast epithelial cell model D492 with single-shot LFQ supported by a SILAC proteomics approach. The D492 EMT cell model contains three cell lines: the epithelial D492 cells, the mesenchymal D492M cells, and a partial mesenchymal, tumorigenic variant of D492 that overexpresses the oncogene HER2. The analysis classified the D492 and D492M cells as basal-like and D492HER2 as claudin-low. Comparative analysis of D492 and D492M to tumorigenic D492HER2 differentiated metabolic markers of migration from those of invasion. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) was one of the top dysregulated enzymes in D492HER2. Gene expression analysis of the cancer genome atlas showed that GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA-mediated knockdown of GFPT2 influenced the EMT marker vimentin and both cell growth and invasion in vitro and was accompanied by lowered metabolic flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide:quinone oxidoreductase (SQOR) in the transsulfuration pathway that regulates H₂S production and mitochondrial homeostasis. Moreover, GFPT2 was within the regulation network of insulin and EGF, and its expression was regulated by reduced glutathione (GSH) and suppressed by the oxidative stress regulator GSK3-β. Our results demonstrate that GFPT2 controls growth and invasion in the D492 EMT model, is a marker for oxidative stress, and associated with poor prognosis in claudin-low breast cancer.     

Integrated Application of Multiomics Strategies Provides Insights Into the Environmental Hypoxia Response in Pelteobagrus vachelli Muscle

Increasing pressures on aquatic ecosystems because of pollutants, nutrient enrichment, and global warming have severely depleted oxygen concentrations. This sudden and significant lack of oxygen has resulted in persistent increases in fish mortality rates. Revealing the molecular mechanism of fish hypoxia adaptation will help researchers to find markers for hypoxia induced by environmental stress. Here, we used a multiomics approach to identify several hypoxia-associated miRNAs, mRNAs, proteins, and metabolites involved in diverse biological pathways in the muscles of Pelteobagrus vachelli. Our findings revealed significant hypoxia-associated changes in muscles over 4 h of hypoxia exposure and discrete tissue-specific patterns. We have previously reported that P. vachelli livers exhibit increased anaerobic glycolysis, heme synthesis, erythropoiesis, and inhibit apoptosis when exposed to hypoxia for 4 h. However, the opposite was observed in muscles. According to our comprehensive analysis, fishes show an acute response to hypoxia, including activation of catabolic pathways to generate more energy, reduction of biosynthesis to decrease energy consumption, and shifting from aerobic to anaerobic metabolic contributions. Also, we found that hypoxia induced muscle dysfunction by impairing mitochondrial function, activating inflammasomes, and apoptosis. The hypoxia-induced mitochondrial dysfunction enhanced oxidative stress, apoptosis, and further triggered interleukin-1β production via inflammasome activation. In turn, interleukin-1β further impaired mitochondrial function or apoptosis by suppressing downstream mitochondrial biosynthesis–related proteins, thus resulting in a vicious cycle of inflammasome activation and mitochondrial dysfunction. Our findings contribute meaningful insights into the molecular mechanisms of hypoxia, and the methods and study design can be utilized across different fish species.