Completed Genome Sequence of the Anaerobic Iron-Oxidizing Bacterium Acidovorax ebreus Strain TPSY

Acidovorax ebreus strain TPSY is the first anaerobic nitrate-dependent Fe(II) oxidizer for which there is a completed genome sequence. Preliminary protein annotation revealed an organism optimized for survival in a complex environmental system. Here, we briefly report the completed and annotated genome sequence of strain TPSY.Microorganisms from diverse anoxic environments are capable of nitrate-dependent Fe(II) oxidation at circumneutral pH (4, 11, 17, 18, 20, 21). Despite their geochemical importance (22), little is known of the underlying biochemical and genetic mechanisms. Genome sequencing of several nitrate-dependent Fe(II) oxidizers will provide insight into this process. By comparing Fe(II) oxidation mechanisms in various organisms, we hope to identify both the conserved and disparate aspects of the metabolism. The genome of Acidovorax ebreus strain TPSY is the first of these to be sequenced.Strain TPSY is a motile, Gram-negative facultative anaerobe isolated from groundwater collected from the U.S. Department of Energy Integrated Field Research Challenge site at Oak Ridge, TN. Growth experiments performed as previously described (21) revealed TPSY''s incapacity for lithoautotrophic growth, which was supported by a lack of genes in the genome encoding any known CO₂ fixation pathways. TPSY did grow mixotrophically with Fe(II) as the electron donor and a 0.1 mM acetate carbon source. 16S rRNA gene sequence analysis placed TPSY in the class Betaproteobacteria with 99.8% similarity to Acidovorax sp. strain JS42 in the family Comamonadaceae.The completed genome consisted of a single circular chromosome of 3,796,573 bp with an average 66.8% G+C content. A total of 3,479 protein-encoding genes were predicted, and 34 (0.98%) had no similarity to public database sequences. Sequencing performed at the Department of Energy Joint Genome Institute (JGI) used Sanger sequencing and 454 pyrosequencing to a depth of 20× coverage. All JGI library construction and sequencing techniques can be found at http://www.jgi.doe.gov/. Sequence assembly, quality assessment, and annotation were performed using the software Phred/Phrap/Consed (www.phrap.com) (6-8), Dupfinisher (10), CRITICA (2), GLIMMER, and GENERATION (5) and the JGI Integrated Microbial Genomes site (12). The completed genome sequence contained 33,341 reads and had an average of ninefold coverage per base and an error rate of <1 in 100,000.TPSY was named in part for its meandering motility, and its genome confirmed the twitching phenotype with the presence of pilT, pilU, and a complete set of flagellar and chemotaxis genes. The ability of TPSY to oxidize simple alcohols and acids with oxygen or nitrate respiration was confirmed by the genome. In addition, biosynthetic pathways for all amino acids except tyrosine and phenylalanine were present. No homologues of chorismate mutase (EC 5.4.99.5), an enzyme required for tyrosine and phenylalanine anabolism, were identified. The genome contained both intact Embden-Meyerhof-Parnas and Entner-Doudoroff pathways, in addition to a pentose phosphate pathway and a trichloroacetic acid cycle.In support of its facultative anaerobicity, a complete set of genes for denitrification and three different terminal oxidases (cytochrome aa₃, cbb₃, and cytochrome d quinol oxidase) were present. The cbb₃ and cytochrome d oxidases, with their high oxygen affinity, putatively enable survival in microaerobic environments (14).TPSY had sequences encoding 30 transposases, 11 integrases, and 11 phage/prophage-related genes. A region of particular interest putatively conferred resistance to lead, arsenate, and mercury: pbrRATARTBC, arsRDAB, and merRPCADE. Evidence suggests horizontal transfer and insertion of this region, as it was flanked on the 5′ end by λ prophage-related genes and the 3′ end encoded a putative Tn21 transposase. Phenotypic studies by the method of Wang et al. (19) revealed MICs of 16 μM phenylmercuric acetate and 250 μM MgCl₂. TPSY was also capable of growth in the presence of arsenate (10 mM) but did not use it as an electron acceptor.Related to phage infection, one CRISPR (clustered, regularly interspaced, short palindromic repeats) region (3, 16) was predicted. The core proteins, the cas1 and cas2 genes, and a csn1 gene formed the CRISPR subtype Nmeni, which is associated with vertebrate pathogens and commensals (9). However, the lack of typical pathogenic type I or III secretion systems such as the hec cluster of Dickeya chrysanthemi (15) or the inv/spa system of Salmonella enterica serovar Typhimurium (13) indicated that TPSY would probably not exhibit a pathogenic lifestyle.     

Expression of a novel tropomyosin isoform in axolotl heart and skeletal muscle

TPM1κ is an alternatively spliced isoform of the TPM1 gene whose specific role in cardiac development and disease is yet to be elucidated. Although mRNA studies have shown TPM1κ expression in axolotl heart and skeletal muscle, it has not been quantified. Also the presence of TPM1κ protein in axolotl heart and skeletal muscle has not been demonstrated. In this study, we quantified TPM1κ mRNA expression in axolotl heart and skeletal muscle. Using a newly developed TPM1κ specific antibody, we demonstrated the expression and incorporation of TPM1κ protein in myofibrils of axolotl heart and skeletal muscle. The results support the potential role of TPM1κ in myofibrillogenesis and sarcomeric function. J. Cell. Biochem. 110: 875–881, 2010. © 2010 Wiley‐Liss, Inc.     

PKC-δ controls the fMLF-induced overproduction of superoxide by neutrophils

Antimicrobial defense by neutrophils implicates the production of reactive oxygen species. Neutrophil responses can be modulated by agonists such as bacterial peptides and proinflammatory factors that modulate neutrophil activity and survival. We evaluated the production of superoxide anions (O₂^?) in response to fMLF by normal human neutrophils after 3 days of preincubation with GM-CSF + IL-4 + TNF-α (survival medium). After 3 days of incubation in survival medium, long-lived neutrophils produced up to six times more O₂^? relative to control neutrophils in response to fMLF and WKYMVM. This augmented response to fMLF was preferentially linked to formyl peptide receptor (FPR), whereas the response to WKYMVM was dependent on formyl peptide receptor-like 1 (FPRL-1). Real-time RT-PCR revealed a diminution of FPR and FPRL-1 expression in neutrophils incubated in survival medium. fMLF-induced overproduction of O₂^? by long-lived neutrophils was independent of intracellular calcium mobilization. The protein tyrosine phosphorylation profile of long-lived neutrophils was modified with respect to control cells. Pharmacological inhibitors of intracellular signals indicated that mechanisms of the excessive fMLF-induced production of O₂^? by long-lived neutrophils implicated the protein kinase C (PKC) pathway, preferentially the PKC-δ isoform, whose protein was augmented in these cells. Thus, long-term cytokine exposure modifies molecular pathways and functional characteristics of the neutrophil.     

Circadian rhythm disruption: health consequences

Circadian rhythms control many facets of biology and their disruption can be associated with severe pathological conditions. In healthy individuals, disturbances to the sleep-wake cycle can be the root cause of many problems. In the present review, we explore the major factors contributing to circadian rhythm disruption, including sleep disorders, jet lag, and shift work. The consequences of these disruptions on central and peripheral clocks and the important areas of the body controlled by them involve immune function and metabolism, as well as alterations in cognitive abilities, thereby impairing social and occupational behavior. Further work exploring clock genes, light exposure, and cellular changes will be critical for identifying how these factors interact to affect health and behavior.     

A note on isolation of spontaneous glucose-resistant mutants of Bacillus subtilis

M ajumdar , S., D as , S.K., B asu , S. & B ose , S.K. 1984. A note on isolation of spontaneous glucose-resistant mutants of Bacillus subtilis. Journal of Applied Bacteriology 56 , 493–494.
The addition of double doses of glucose delayed the initiation of sporulation in Bacillus subtilis B₃₄ by 42 h. Heat-treatment of the culture within the delay period, (selectively killing the vegetative cells and leaving the spores unaffected) followed by immediate plating, afforded a good method for isolating spontaneous glucose-resistant sporulation mutants.     

Non-specific phospholipases C2 and C6 redundantly function in pollen tube growth via triacylglycerol production in Arabidopsis

Non-specific phospholipase Cs (NPCs) are responsible for membrane lipid remodeling that involves hydrolysis of the polar head group of membrane phospholipids. Arabidopsis NPC2 and NPC6 are essential in gametogenesis, but their underlying role in the lipid remodeling remains elusive. Here, we show that these NPCs are required for triacylglycerol (TAG) production in pollen tube growth. NPC2 and NPC6 are highly expressed in developing pollen tubes and are localized at the endoplasmic reticulum. Mutants of NPC2 and NPC6 showed reduced rate of pollen germination, length of pollen tube and amount of lipid droplets (LDs). Overexpression of NPC2 or NPC6 induced LD accumulation, which suggests that these NPCs are involved in LD production. Furthermore, mutants defective in the biosynthesis of TAG, a major component of LDs, showed defective pollen tube growth. These results suggest that NPC2 and NPC6 are essential in gametogenesis for a role in hydrolyzing phospholipids and producing TAG required for pollen tube growth. Thus, lipid remodeling from phospholipids to TAG during pollen tube growth represents an emerging role for the NPC family in plant developmental control.     

An approach towards understanding the genesis of sunlight-induced skin cancer

The molecular basis of the sunlight-induced skin carcinogenesis has been elucidated. Of the two ultraviolet components of sunlight that reach the earth's surface the UV-B is known to be carcinogenic but the mode of action of UV-A, the predominant component of sunlight, is ill understood. Using the liposomes as a model system, it has been shown here that UV-A causes dose-dependent lipid peroxidation as estimated by measurements of conjugated dienes, lipid hydroperoxides, malondialdehydes and the fluorescent adducts (Schiff bases) produced by the reaction of MDA with glycine. Direct exposure to sunlight has also been shown to cause dose-dependent lipid peroxidation. The UV-A induced lipid peroxidation has also been shown to be dependent on dose rate. While the sodium formate, dimethyl sulphoxide, superoxide dismutase and EDTA do not have any significant effect, sodium azide, histidine, beta-carotene and dimethylfuran were shown to inhibit significantly the UV-A induced lipid peroxidation, thereby providing significant evidence of the involvement of singlet oxygen (1O2) as the initiating agent. The use of D2O in place of H2O as the liposome dispersing medium enhanced to great extent the UV-A induced lipid peroxidation, thereby lending additional support to the finding that singlet oxygen was the initiating agent. The possible mode of formation of 1O2 on exposure to UV-A was discussed. This study also highlighted the role of environmental factors on the sunlight-induced cutaneous damage. Finally, the relation between lipid peroxidation, DNA damage and carcinogenesis has been discussed in a way to suggest the possible link between sunlight exposure and causation of skin cancer.