Whole genome sequence and comparative genomics analysis of multidrug-resistant Staphylococcus xylosus NM36 isolated from a cow with mastitis in Basrah city.

BACKGROUND: Staphylococcus xylosus is a coagulase-negative, gram-positive coccus that is found in the environment and as a commensal organism on the skin and mucosal surfaces of animals. Despite the fact that S. xylosus is considered a nonpathogenic bacterium, several studies have linked S. xylosus to opportunistic infections in both animals and humans. During an investigation of mastitis-causing agents in the governorate of Basrah, Iraq, we identified an antibiotic-resistant strain of S. xylosus NM36 from a milk sample from a cow with chronic mastitis. In addition to robust biofilm formation, multiple antibiotic resistance phenotypes were found. To further understand the genetic background for these phenotypes, the full genome of S. xylosus NM36 was analyzed. RESULTS: The genome consisted of a single circular 2,668,086 base pairs chromosome containing 32.8% G + C. There were 2454 protein-coding sequences, 4 ribosomal RNA (rRNA) genes, and 50 transfer RNA (tRNA) genes in the genome. In addition, genetic variation was studied by searching sequence data against a representative reference genome. Consequently, single-nucleotide polymorphism analysis was conducted and showed that there were 46,610 single-nucleotide polymorphisms (SNPs), 523 insertions, and 551 deletions. In order to overcome antibiotics, S. xylosus NM36 had been armed with several antibiotic resistance genes from several groups and families. The genome annotation service in PathoSystems Resource Integration Center (PATRIC) and Rapid Annotation using Subsystem Technology (RAST) annotation servers showed that there are multiple antimicrobial resistance elements, including antibiotic inactivation enzymes (BlaZ family, FosB), antibiotic resistance gene clusters (TcaB, TcaB2, TcaR), proteins involved in methicillin resistance (LytH, FmtA, FemC, HmrB, HmrA), TetR family transcriptional regulators, and efflux pumps conferring antibiotic resistance (NorA). In addition, we investigated and categorized the biofilm and quorum-sensing elements of the NM36 strain and found that it has multiple subsets of biofilm regulators, confirming its pathogenic nature. CONCLUSIONS: These findings necessitate a reevaluation of microbial and clinical interventions when dealing with coagulase-negative staphylococci, particularly in the context of studies pertaining to public health. This is the first time, to our knowledge, that the entire genome of S. xylosus has been sequenced in Iraq.

Two NAD-linked redox shuttles maintain the peroxisomal redox balance in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, peroxisomes are the sole site of fatty acid ß-oxidation. During this process, NAD+ is reduced to NADH. When cells are grown on oleate medium, peroxisomal NADH is reoxidised to NAD+ by malate dehydrogenase (Mdh3p) and reduction equivalents are transferred to the cytosol by the malate/oxaloacetate shuttle. The ultimate step in lysine biosynthesis, the NAD+-dependent dehydrogenation of saccharopine to lysine, is another NAD+-dependent reaction performed inside peroxisomes. We have found that in glucose grown cells, both the malate/oxaloacetate shuttle and a glycerol-3-phosphate dehydrogenase 1(Gpd1p)-dependent shuttle are able to maintain the intraperoxisomal redox balance. Single mutants in MDH3 or GPD1 grow on lysine-deficient medium, but an mdh3/gpd1Δ double mutant accumulates saccharopine and displays lysine bradytrophy. Lysine biosynthesis is restored when saccharopine dehydrogenase is mislocalised to the cytosol in mdh3/gpd1Δ cells. We conclude that the availability of intraperoxisomal NAD+ required for saccharopine dehydrogenase activity can be sustained by both shuttles. The extent to which each of these shuttles contributes to the intraperoxisomal redox balance may depend on the growth medium. We propose that the presence of multiple peroxisomal redox shuttles allows eukaryotic cells to maintain the peroxisomal redox status under different metabolic conditions.

Pnc1 piggy-back import into peroxisomes relies on Gpd1 homodimerisation.

Peroxisomes are eukaryotic organelles that posttranslationally import proteins via one of two conserved peroxisomal targeting signal (PTS1 or 2) mediated pathways. Oligomeric proteins can be imported via these pathways but evidence is accumulating that at least some PTS1-containing monomers enter peroxisomes before they assemble into oligomers. Some proteins lacking a PTS are imported by piggy-backing onto PTS-containing proteins. One of these proteins is the nicotinamidase Pnc1, that is co-imported with the PTS2-containing enzyme Glycerol-3-phosphate dehydrogenase 1, Gpd1. Here we show that Pnc1 co-import requires Gpd1 to form homodimers. A mutation that interferes with Gpd1 homodimerisation does not prevent Gpd1 import but prevents Pnc1 co-import. A suppressor mutation that restores Gpd1 homodimerisation also restores Pnc1 co-import. In line with this, Pnc1 interacts with Gpd1 in vivo only when Gpd1 can form dimers. Redirection of Gpd1 from the PTS2 import pathway to the PTS1 import pathway supports Gpd1 monomer import but not Gpd1 homodimer import and Pnc1 co-import. Our results support a model whereby Gpd1 may be imported as a monomer or a dimer but only the Gpd1 dimer facilitates co-transport of Pnc1 into peroxisomes.