Characterization and whole-genome sequencing of an extreme arsenic-tolerant Citrobacter freundii SRS1 strain isolated from Savar area in Bangladesh.

Citrobacter freundii SRS1, gram-negative bacteria, were isolated from Savar, Bangladesh. The strain could tolerate up to 80 mmol L-1 sodium arsenite, 400 mmol L-1 sodium arsenate, 5 mmol L-1 manganese sulfate, 3 mmol L-1 lead nitrate, 2.5 mmol L-1 cobalt chloride, 2.5 mmol L-1 cadmium acetate, and 2.5 mmol L-1 chromium chloride. The whole-genome sequencing revealed that the genome size of C. freundii SRS1 is estimated to be 5.4 Mb long, and the G + C content is 51.7%. The genome of C. freundii SRS1 contains arsA, arsB, arsC, arsD, arsH, arsR, and acr3 genes for arsenic resistance; czcA, czcD, cbiN, and cbiM genes for cobalt resistance; chrA and chrB genes for chromium resistance; mntH, sitA, sitB, sitC, and sitD genes for manganese resistance; and zntA gene for lead and cadmium resistance. This novel acr3 gene has never previously been reported in any C. freundii strain except SRS1. A set of 130 completely sequenced strains of C. freundii was selected for phylogenomic analysis. The phylogenetic tree showed that the SRS1 strain is closely related to the C. freundii 62 strain. Further analyses of the genes involved in metal and metalloid resistance might facilitate identifying the mechanisms and pathways involved in high metal resistance in the C. freundii SRS1 strain.

Whole-genome sequencing and comparative analysis of heavy metals tolerant Bacillus anthracis FHq strain isolated from tannery effluents in Bangladesh.

Heavy metal contamination of the environment is a primary concern in Bangladesh. This study aims to characterize a novel heavy metal tolerant strain, Bacillus anthracis FHq, isolated from the tannery effluents of Savar, Bangladesh. The strain could tolerate up to 5 mM of lead nitrate, 2.5 mM of sodium arsenate, chromium chloride, cobalt chloride, 1.5 mM cadmium acetate, and 1 mM of sodium arsenite. Whole-genome sequencing analysis revealed that the genome of the strain is around 5.2 Mbp long, and the G + C content is 35.4%. Besides, FHq has genes cadC, zntA, arsCR, czcD, and chrA, which confer lead, arsenic, cobalt, and chromium resistance, respectively. A total of nineteen other closely related and completely sequenced B. anthracis strains were selected based on average nucleotide identity along with the FHq strain for phylogenomic and pan-genome analysis. The phylogenomic analysis predicted the inter-genomic evolutionary relationship of the strain isolated from Bangladesh, and it was closely related to a strain isolated from China. Pan-genome analysis revealed that the FHq strain possesses 6045 pan genes, 3802 core genes, and 152 unique genes in its genomic content. Hence, the genetic information and comparative analysis of the FHq strain might facilitate identifying the mechanisms conferring high resistance to lead in B. anthracis strains isolated from Bangladesh.

Proteomic Shifts Reflecting Oxidative Stress and Reduced Capacity for Protein Synthesis, and Alterations to Mitochondrial Membranes in Neurospora crassa Lacking VDAC.

Voltage-dependent anion-selective channels (VDAC) maintain the bidirectional flow of small metabolites across the mitochondrial outer membrane and participate in the regulation of multiple cellular processes. To understand the roles of VDAC in cellular homeostasis, preliminary proteomic analyses of S100 cytosolic and mitochondria-enriched fractions from a VDAC-less Neurospora crassa strain (ΔPor-1) were performed. In the variant cells, less abundant proteins include subunits of translation initiation factor eIF-2, enzymes in the shikimate pathway leading to precursors of aromatic amino acids, and enzymes involved in sulfate assimilation and in the synthesis of methionine, cysteine, alanine, serine, and threonine. In contrast, some of the more abundant proteins are involved in electron flow, such as the α subunit of the electron transfer flavoprotein and lactate dehydrogenase, which is involved in one pathway leading to pyruvate synthesis. Increased levels of catalase and catalase activity support predicted increased levels of oxidative stress in ΔPor-1 cells, and higher levels of protein disulfide isomerase suggest activation of the unfolded protein response in the endoplasmic reticulum. ΔPor-1 cells are cold-sensitive, which led us to investigate the impact of the absence of VDAC on several mitochondrial membrane characteristics. Mitochondrial membranes in ΔPor-1 are more fluid than those of wild-type cells, the ratio of C18:1 to C18:3n3 acyl chains is reduced, and ergosterol levels are lower. In summary, these initial results indicate that VDAC-less N. crassa cells are characterized by a lower abundance of proteins involved in amino acid and protein synthesis and by increases in some associated with pyruvate metabolism and stress responses. Membrane lipids and hyphal morphology are also impacted by the absence of VDAC.

Increased reactive oxygen species production and maintenance of membrane potential in VDAC-less Neurospora crassa mitochondria.

The highly abundant voltage-dependent anion-selective channel (VDAC) allows transit of metabolites across the mitochondrial outer membrane. Previous studies in Neurospora crassa showed that the LoPo strain, expressing 50% of normal VDAC levels, is indistinguishable from wild-type (WT). In contrast, the absence of VDAC (ΔPor-1), or the expression of an N-terminally truncated variant VDAC (ΔN2-12porin), is associated with deficiencies in cytochromes b and aa3 of complexes III and IV and concomitantly increased alternative oxidase (AOX) activity. These observations led us to investigate complex I and complex II activities in these strains, and to explore their mitochondrial bioenergetics. The current study reveals that the total NADH dehydrogenase activity is similar in mitochondria from WT, LoPo, ΔPor-1 and ΔN2-12porin strains; however, in ΔPor-1 most of this activity is the product of rotenone-insensitive alternative NADH dehydrogenases. Unexpectedly, LoPo mitochondria have increased complex II activity. In all mitochondrial types analyzed, oxygen consumption is higher in the presence of the complex II substrate succinate, than with the NADH-linked (complex I) substrates glutamate and malate. When driven by a combination of complex I and II substrates, membrane potentials (Δψ) and oxygen consumption rates (OCR) under non-phosphorylating conditions are similar in all mitochondria. However, as expected, the induction of state 3 (phosphorylating) conditions in ΔPor-1 mitochondria is associated with smaller but significant increases in OCR and smaller decreases in Δψ than those seen in wild-type mitochondria. High ROS production, particularly in the presence of rotenone, was observed under non-phosphorylating conditions in the ΔPor-1 mitochondria. Thus, the absence of VDAC is associated with increased ROS production, in spite of AOX activity and wild-type OCR in ΔPor-1 mitochondria.

In silico analysis of coevolution among ERMES proteins, Pex11, and Lam6.

In eukaryotic cells, communication and dynamic interactions among different organelles are important for maintaining cellular homeostasis. The endoplasmic reticulum (ER) mitochondria encounter structure (ERMES) complex establishes membrane contact sites between ER and mitochondria and is essential for phospholipid transport, protein import, and mitochondrial dynamics and inheritance. In this work, in silico analyses were used to probe the intramolecular interactions in ERMES proteins and the interactions that support the ERMES complex. Based on mutual information (MI), sites of intramolecular coevolution are predicted in the core proteins Mmm1, Mdm10, Mdm12, Mdm34, the peroxisomal protein Pex11, and cytoplasmic Lam6; these sites are linked to structural features of the proteins. Intermolecular coevolution is predicted among the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domains of Mmm1, Mdm12, and Mdm34. Segments of Pex11 and Lam6 also share MI with the SMP domains of Mmm1 and Mdm12 and with the N terminus of Mdm34, implicating Mdm34 as part of a hub for interactions between ERMES and other complexes. In contrast, evidence of limited intermolecular coevolution involving the outer membrane protein Mdm10 was detected only with Mmm1 and Pex11. The results support models for the organization of these interacting proteins and suggest roles for Pex11 and Lam6 in regulating complex formation.

Functional characterization of an N-terminally-truncated mitochondrial porin expressed in Neurospora crassa.

Mitochondrial porin, which forms voltage-dependent anion-selective channels (VDAC) in the outer membrane, can be folded into a 19-ß-stranded barrel. The N terminus of the protein is external to the barrel and contains α-helical structure. Targeted modifications of the N-terminal region have been assessed in artificial membranes, leading to different models for gating in vitro. However, the in vivo requirements for gating and the N-terminal segment of porin are less well-understood. Using Neurospora crassa porin as a model, the effects of a partial deletion of the N-terminal segment were investigated. The protein, ΔN2-12porin, is assembled into the outer membrane, albeit at lower levels than the wild-type protein. The resulting strain displays electron transport chain deficiencies, concomitant expression of alternative oxidase, and decreased growth rates. Nonetheless, its mitochondrial genome does not contain any significant mutations. Most of the genes that are expressed in high levels in porin-less N. crassa are expressed at levels similar to that of wild type or are slightly increased in ΔN2-12porin strains. Thus, although the N-terminal segment of VDAC is required for complete function in vivo, low levels of a protein lacking part of the N terminus are able to rescue some of the defects associated with the absence of porin.

The N-terminus of VDAC: Structure, mutational analysis, and a potential role in regulating barrel shape.

A novel feature of the voltage-dependent anion channel (VDAC, mitochondrial porin), is the barrel, comprising an odd number of ß-strands and closed by parallel strands. Recent research has focused on the N-terminal segment, which in the available structures, resides in the lumen and is not part of the barrel. In this review, the structural data obtained from vertebrate VDAC are integrated with those from VDAC in artificial bilayers, emphasizing the array of native and tagged versions of VDAC used. The data are discussed with respect to a recent gating model (Zachariae et al. (2012) Structure 20:1-10), in which the N-terminus acts not as a gate on a stable barrel, but rather stabilizes the barrel, preventing its shift into a partially collapsed, low-conductance, closed state. Additionally, the role of the N-terminus in VDAC oligomerization, apoptosis through interactions with hexokinase and its interaction with ATP are discussed briefly.