text:
- Research article
- Open Access
The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence
- Eva Trost1, 2,
- Lisa Ott3,
- Jessica Schneider1, 2, 4,
- Jasmin Schröder1,
- Sebastian Jaenicke4,
- Alexander Goesmann4,
- Peter Husemann5,
- Jens Stoye5,
- Fernanda Alves Dorella6,
- Flavia Souza Rocha6,
- Siomar de Castro Soares6,
- Vívian D'Afonseca6,
- Anderson Miyoshi6,
- Jeronimo Ruiz7,
- Artur Silva8,
- Vasco Azevedo6,
- Andreas Burkovski3,
- Nicole Guiso9,
- Olivier F Join-Lambert10,
- Samer Kayal11 and
- Andreas Tauch1Email author
© Trost et al; licensee BioMed Central Ltd. 2010
- Received: 8 September 2010
- Accepted: 30 December 2010
- Published: 30 December 2010
Corynebacterium pseudotuberculosis is generally regarded as an important animal pathogen that rarely infects humans. Clinical strains are occasionally recovered from human cases of lymphadenitis, such as C. pseudotuberculosis FRC41 that was isolated from the inguinal lymph node of a 12-year-old girl with necrotizing lymphadenitis. To detect potential virulence factors and corresponding gene-regulatory networks in this human isolate, the genome sequence of C. pseudotuberculosis FCR41 was determined by pyrosequencing and functionally annotated.
Sequencing and assembly of the C. pseudotuberculosis FRC41 genome yielded a circular chromosome with a size of 2,337,913 bp and a mean G+C content of 52.2%. Specific gene sets associated with iron and zinc homeostasis were detected among the 2,110 predicted protein-coding regions and integrated into a gene-regulatory network that is linked with both the central metabolism and the oxidative stress response of FRC41. Two gene clusters encode proteins involved in the sortase-mediated polymerization of adhesive pili that can probably mediate the adherence to host tissue to facilitate additional ligand-receptor interactions and the delivery of virulence factors. The prominent virulence factors phospholipase D (Pld) and corynebacterial protease CP40 are encoded in the genome of this human isolate. The genome annotation revealed additional serine proteases, neuraminidase H, nitric oxide reductase, an invasion-associated protein, and acyl-CoA carboxylase subunits involved in mycolic acid biosynthesis as potential virulence factors. The cAMP-sensing transcription regulator GlxR plays a key role in controlling the expression of several genes contributing to virulence.
The functional data deduced from the genome sequencing and the extended knowledge of virulence factors indicate that the human isolate C. pseudotuberculosis FRC41 is equipped with a distinct gene set promoting its survival under unfavorable environmental conditions encountered in the mammalian host.
- Nonribosomal Peptide Synthetase
- Potential Virulence Factor
- Siderophore Biosynthesis
- Caseous Lymphadenitis
- Pilin Subunit
Corynebacterium pseudotuberculosis is generally regarded as an important animal pathogen and the etiological agent of a disease that is commonly called caseous lymphadenitis [1, 2]. This bacterium is predominantly isolated from sheep and goats (biovar ovis), but has been recognized also in other animals, including horses and cattle (biovar equi) [1]. The importance of caseous lymphadenitis varies greatly around the world, but this disease is found in all major sheep and goat production areas [2]. C. pseudotuberculosis is a significant cause of morbidity in sheep and goats, and caseous lymphadenitis in these animals resulted in economic losses, for instance in wool, milk and meat production [2, 3]. C. pseudotuberculosis is a facultative intracellular pathogen that is able to survive and grow in macrophages, thus escaping the immune response of the host [1, 4]. A close phylogenetic relationship between C. pseudotuberculosis and Corynebacterium ulcerans was proposed as both species are unique among the corynebacteria in producing phospholipase D. The sphingomyelin-degrading enzyme is regarded as the major virulence factor for C. pseudotuberculosis[5, 6]. This exotoxin promotes the hydrolysis of ester bonds in sphingomyelin in mammalian cell membranes and contributes to the spread of the bacterium from the initial site of infection to secondary sites within the host.
Although infections due to C. pseudotuberculosis are predominantly observed in sheep and goats, infections due to this pathogen also occur in humans [7, 8]. The number of human infections is rare, but it might be underestimated as corynebacteria are often considered as skin contaminants in clinical specimens [9]. Published cases of human infections by C. pseudotuberculosis usually presented as suppurative lymphadenitis [7, 8], with the exception of one case of eosinophilic pneumonia [10]. Most patients revealed a classical risk exposure of close contact with animals, in particular with sheep. C. pseudotuberculosis infects humans via superficial wounds, forming abscesses in the regional draining lymph nodes after an incubation period ranging from weeks to months. In most of the published cases, antibiotic treatment alone was unsuccessful and the antimicrobial therapy was therefore supplemented by surgical interventions [7, 8]. The general problem in achieving an effective treatment of C. pseudotuberculosis infections in humans and animals is probably related to the facultative intracellular lifestyle of this bacterium, as it can survive and multiply in macrophages [4]. The cell death and subsequent release of the pathogen lead to necrotic lesions and the formation of thick collagen capsules that cannot be penetrated by antibiotics [11].
In the present study, we characterize the genome of C. pseudotuberculosis FRC41 that was isolated from the inguinal lymph node of a 12-year-old French girl with necrotizing lymphadenitis [12]. This patient had no underlying disease or predisposing conditions. The diagnosis of necrotizing lymphadenitis was supported by 16 S rDNA gene sequencing, a taxonomic classification of the bacterial isolate and Western blot assays revealing the presence of phospholipase D in the patient's serum. The patient relapsed many times despite a surgical drainage and an adapted antimicrobial treatment, although C. pseudotuberculosis FRC41 turned out to be highly susceptible to a wide range of antibiotics in vitro. However, the patient recovered after a broad spectrum intravenous antimicrobial therapy with imipenem-cilastatin, rifampin and ofloxacin for four months, followed by an oral therapy with rifampin and ofloxacin for six months [12]. Here, we report the functional annotation of the complete genome sequence of C. pseudotuberculosis FRC41, the detection of potential virulence factors and the deduced gene-regulatory networks controlling their expression.
Results and discussion
Pyrosequencing and annotation of the C. pseudotuberculosis FRC41 genome
The annotation of the C. pseudotuberculosis FRC41 genome sequence was performed with the GenDB software system [15] and resulted in the detection of 2,110 protein-coding regions. Furthermore, 49 tRNA genes were predicted by the tRNAscan-SE program [16] and four rrn operons were detected on the leading strands of the chromosome (Figure 1B). A plot of the calculated G/C skew [(G-C)/(G+C)] indicated a bi-directional replication mechanism of the C. pseudotuberculosis chromosome (Figure 1A). According to the presence and distribution of six conserved DnaA boxes, the oriC is located downstream of the dnaA coding region [17]. The G/C skew and the biased distribution of architecture imparting sequences (AIMS) on the leading and lagging strands indicated the presence of a dif region [18] at the expected position of 180° from oriC, dividing the chromosome of C. pseudotuberculosis FRC41 into two replichores of similar size (Figure 1B). Synteny analysis by reciprocal best matches with BLASTP [19] revealed a highly conserved order of orthologous genes between the chromosomes of C. pseudotuberculosis FRC41 and C. diphtheriae NCTC 13129 (Figure 1C), which is consistent with the close phylogenetic relationship of both species [1] and the observation that genetic rearrangements are rare in the genomes of species belonging to the main lineage of the genus Corynebacterium[20, 21]. The calculated reciprocal best BLASTP hits [19] were used also to compare the predicted proteome of C. pseudotuberculosis FRC41 with the complete set of proteins encoded in the genome of C. diphtheriae NCTC 13129 [14]. This comparative content analysis at the proteome level revealed that 1610 proteins (76.3%) of C. pseudotuberculosis FRC41 share a homologue in the genome of C. diphtheriae NCTC 13129 (data not shown). The characteristic features of C. pseudotuberculosis FRC41 are apparently based on a distinct gene set, defining its lifestyle and pathogenicity, such as the pld gene encoding phospholipase D [22]. In the following sections, we describe a collection of relevant genes contributing to the lifestyle and pathogenicity of C. pseudotuberculosis FRC41 and deduce their integration into a transcriptional gene-regulatory network.
The transcriptional regulatory repertoire of C. pseudotuberculosis FRC41
Iron regulation in physiology and virulence of C. pseudotuberculosis
The dtxR gene of C. pseudotuberculosis FRC41 encodes a homologue of the diphtheria toxin repressor DtxR that is activated by iron and controls a complex gene-regulatory network involved in iron homeostasis in corynebacteria [30, 31]. The detection of a putative DtxR-binding site in the fagA-fagD intergenic region supports the previously observed differential expression pattern of the fagABC operon in vitro[29]. We combined the functional annotation of the C. pseudotuberculosis FRC41 genome sequence with a bioinformatic motif search for DtxR-binding sites using a hidden Markov model and a position weight matrix with input data from the DtxR regulon of C. glutamicum[31]. In this way, additional genes were assigned to the DtxR regulon of C. pseudotuberculosis FRC41, including several gene clusters involved in the utilization of various host compounds as iron sources (Figure 3). One gene region revealed similarity to the hemin utilization system HmuTUV from C. diphtheriae[32]. The corresponding hemin binding protein HtaA is probably associated with the cell envelope and involved in the utilization of heme iron [33]. Two gene clusters assigned to the DtxR regulon of C. pseudotuberculosis FRC41 include htaA-like genes (htaD and htaF) that are associated with genes encoding membrane proteins (htaE and htaG), suggesting a role of these clusters in the acquisition of iron from the host (Figure 3). The hmuO gene of the DtxR regulon encodes heme oxygenase that releases iron from the protoporphyrin ring of heme and facilitates the acquisition of iron from heme and hemoglobin [34]. Because of the potential toxicity of both, iron and heme, the expression of hmuO in C. diphtheriae is under complex control, comprising the iron-responsive repressor DtxR and the heme-dependent activators ChrA and HrrA that are part of the two-component signal transduction systems ChrA-ChrS and HrrA-HrrS [35, 36]. A BLAST search across the C. pseudotuberculosis FRC41 genome revealed two response regulators that share similarity with ChrA and HrrA. As the hrrA gene of C. pseudotuberculosis FRC41 is part of the DtxR regulon, a complex hierarchical control of hmuO gene expression might be established in this bacterium (Figure 3).
Iron acquisition can moreover involve the synthesis and secretion of high-affinity iron chelators, termed siderophores, which are synthesized by nonribosomal peptide synthetases or by biosynthesis pathways independent of these multimodular enzymes [37, 38]. The genome of C. pseudotuberculosis FRC41 contains two DtxR-regulated gene clusters that are probably associated with pathways for siderophore biosynthesis independent of nonribosomal peptide synthetases (Figure 3). The ciu locus comprises the ciuABCD (ABC-type transporter), ciuE (siderophore biosynthesis-related protein) and ciuF (putative efflux protein) genes. The predicted product of the ciuE gene is similar to aerobactin biosynthesis enzymes [39]. The expression of the ciuA gene encoding the lipoprotein receptor of the ABC transport system has been detected in vivo by a reporter transposon system [40]. The ciu gene region detected in the genome of C. pseudotuberculosis FRC41 is similar to the ciu gene locus from C. diphtheriae NCTC 13129, with the exception that it lacks the ciuG gene, encoding a protein of unknown function [41].
The second DtxR-regulated gene cluster related to siderophore biosynthesis and excretion includes four genes probably constituting the biosynthesis pathway (ogs, ocd, odc and tsb), a gene encoding an efflux protein (mdtK) and four genes encoding an ABC-type transporter (stsABCD) (Figure 3). Ornithine cyclodeaminase (ocd) and ornithine decarboxylase (odc) as well as monooxygenase (ogs) and synthetase (tsb) functions encoded in this gene cluster are components of widely distributed routes for siderophore biosynthesis [39]. Additional DtxR-binding sites were detected in front of ftn (ferritin) and fas (fatty acid synthase) and the sdhCAB (succinate dehydrogenase) operon that are also part of the DtxR regulon in C. glutamicum[31, 42]. Ferritins act primarily in iron storage and are thus central to the natural regulation of iron in the bacterial cell [28].
Furthermore, the ripA gene encoding a DNA-binding transcription regulator of the AraC protein family was assigned to the DtxR regulon of C. pseudotuberculosis FRC41 (Figure 3). The expression of the orthologous ripA gene from C. glutamicum is also controlled directly by DtxR [42]. Under iron limitation, the RipA protein acts as a repressor of genes coding for iron proteins in C. glutamicum[42]. Candidate RipA-binding sites were detected by bioinformatic pattern searches in the genome sequence of C. pseudotuberculosis FRC41 in front of the acn (aconitase) gene and upstream of the sdhCAB (succinate dehydrogenase) and cydABDC operons (cytochrome bd-type menaquinol oxidase and ABC-type transporter), thereby linking the availability of iron with the expression of genes in the citrate cycle and the respiratory energy metabolism of C. pseudotuberculosis. Due to its high oxygen affinity, the cytochrome bd oxidase is used in many bacteria under microaerobic growth conditions [43]. Additional RipA-binding sites were detected in front of the katA (catalase) gene and in the dps-fpg1 (DNA protection during starvation protein, formamidopyrimidine-DNA glycosylase) intergenic region, integrating protective functions into the RipA regulon (Figure 3). Dps-like proteins effectively protect DNA against oxidizing agents by nonspecific DNA-binding and physical sequestration that limits DNA accessibility to detrimental factors [44]. They also act as iron-binding and storage proteins and catalyze the oxidation of ferrous iron to ferric iron by H2O2, which prevents the formation of hydroxyl radicals by the Fenton reaction [45]. The lsr2 gene of C. pseudotuberculosis FRC41 encodes the multifunctional histone-like protein and transcription regulator Lsr2 (Figure 2) that shares a number of physical properties with Dps-like proteins and the ability to bind DNA sequences with little specificity [46]. Like Dps, the Lsr2 protein may physically protect corynebacterial DNA against reactive oxygen intermediates [47]. Formamidopyrimidine-DNA glycosylase is a primary participant in the repair of DNA lesions caused by oxidative damage [48]. A second gene (fpg2) encoding formamidopyrimidine-DNA glycosylase was detected in the genome of C. pseudotuberculosis FRC41, but the upstream region of fpg2 apparently lacks a RipA-binding site. Catalase is another important protective component in the bacterial oxidative stress response which is involved in the detoxification of H2O2[49]. Hence, the gene composition of the DtxR-RipA gene-regulatory network revealed the interdependence of iron metabolism and oxidative stress response and the regulatory connection between distinct physiological functionalities of the corynebacterial cell, including an important role in virulence of C. pseudotuberculosis.
Manganese and zinc regulation in physiology and virulence of C. pseudotuberculosis
As manganese and zinc ions are apparently involved as cofactors in the oxidative stress response of pathogens, the corresponding regulons involved in metal ion uptake were identified in the genome sequence of C. pseudotuberculosis FRC41 (Figure 4B). The uptake of manganese in C. pseudotuberculosis FRC41 is mediated by a typical manganese ABC-type transport system (mntABCD) that is negatively controlled at the transcriptional level by the metalloregulator MntR (Figure 4B). The manganese ABC transporter is composed of: a lipoprotein receptor (MntA), anchored to the cell membrane and functioning as an extracellular cation-binding protein; a cytoplasmic ATP-binding protein (MntB); and two integral membrane proteins (MntC and MntD) that mediate the cation flux [63]. A homologous gene cluster and its manganese-dependent transcriptional control by the metalloregulator MntR have been examined in the genome of C. diphtheriae[64]. The uptake of zinc ions in C. pseudotuberculosis FRC41 is probably mediated by two ABC transport systems (znuB1C1A1 and znuB2A2C2), as both gene regions are specified by the presence of Zur-binding sites (Figure 4C). The Zur protein is a metalloregulator of the ferric uptake regulator (FUR) family of DNA-binding transcription regulators [65, 66]. The znu ABC transporter genes are key components of actinobacterial Zur regulons and their expression is generally repressed by Zur in a zinc-dependent manner [66]. Zinc resistance might be facilitated in C. pseudotuberculosis FRC41 by the ArsR-type transcription regulator Znr that probably controls the expression of the czcE gene encoding a cobalt/zinc/cadmium efflux system [23, 67, 68]. The yciC gene coding for a putative P-loop GTPase of the COG0523 protein family is also part of the Zur regulon in C. pseudotuberculosis FRC41 (Figure 4C). The YciC protein may function as a metallochaperone/insertase to enable the in vivo assembly of zinc-containing proteins under environmental conditions of zinc deficiency [69].
Moreover, the genome-wide motif search for Zur-binding sites in C. pseudotuberculosis FRC41 detected three genes (cpfrc_00168, cpfrc_00313, cpfrc_02059) encoding proteins with a carboxy-terminal sorting (LPxTG) signal that is generally used by Gram-positive bacteria to anchor proteins to the cell wall [70]. The Cpfrc_00168 protein contains two CnaB-like domains that may be involved in the positioning of a ligand binding domain away from the corynebacterial cell surface and is encoded adjacent to a putative sialoprotein-binding protein. The secreted proteins Cpfrc_00313 and Cpfrc_02059 contain actinobacterial surface-anchored protein domains for their covalent attachment to the cell wall [56]. The Cpfrc_00313 protein is encoded next to the components of a transporter and may act together with the Cpfrc_00314 protein as a substrate receptor for this system. Hence, the reconstruction of regulons participating in metal ion uptake of C. pseudotuberculosis FRC41 led to the detection of genes that may fulfill novel functions in sensing the presence of zinc in the environment.
Genes encoding adhesive pili in C. pseudotuberculosis FRC41
Candidate virulence factors of C. pseudotuberculosis FRC41 and their integration into a gene-regulatory network
Candidate determinants contributing to virulence of C. pseudotuberculosis FRC41
Predicted protein function
phospholipase D (sphingomyelin-degrading enzyme)
corynebacterial protease CP40 (serine protease)
secreted subtilisin-like serine protease
secreted subtilisin-like serine protease
secreted trypsin-like serine protease
secreted SGNH-hydrolase
neuraminidase H (sialidase)
resuscitation-promoting factor interacting protein (D,L-endopeptidase)
resuscitation-promoting factor A (muralytic enzyme)
resuscitation-promoting factor B (muralytic enzyme)
nitric oxide reductase
nonribosomal peptide synthetase 1
nonribosomal peptide synthetase 2
acetyl-CoA carboxylase β-subunit involved in fatty acid synthesis
acyl-CoA carboxylase β-subunit involved in mycolic acid synthesis
acyl-CoA carboxylase β-subunit involved in mycolic acid synthesis
Likewise, a DNA-binding site for GlxR was detected in front of the rpfI gene encoding an invasion-associated protein that is involved in cell surface organization and adhesion of corynebacteria [86]. The homologue of RpfI in M. tuberculosis (named RipA) revealed endopeptidase activity and interacts with the resuscitation-promoting factor RpfB, representing a peptidoglycan glycosidase [86, 87]. Two genes (rpfA and rpfB) encoding resuscitation-promoting factors are present in the genome of C. pseudotuberculosis FRC41 (Table 1). Important roles in pathogenesis for peptidoglycan hydrolytic enzymes have been proposed [88] and an analogous system combining the activities of a muramidase and an endopeptidase contributed to the virulence of Listeria monocytogenes[89]. As previously demonstrated in C. glutamicum[74, 90], the expression of rpfI, rpfA and rpfB in C. pseudotuberculosis FRC41 is probably under complex control by three regulatory proteins, GlxR, RamB and RamA (Figure 6).
Another potential virulence factor of C. pseudotuberculosis FRC41 is represented by the nor gene encoding nitric oxide reductase (Table 1). This enzyme is generally involved in the detoxification of nitric oxide and consequently necessary for the long-term persistence of pathogens in macrophages [91]. The toxic properties of nitric oxide are used by the host immune system to kill or slow down the growth of pathogenic bacteria [51]. Interestingly, the expression of the nor gene was not induced upon the infection of macrophages by animal C. pseudotuberculosis[4]. As the expression of nor is typically activated by a transcription regulator in response to the presence of nitric oxide [92], the regulatory pattern of nor transcription and its contribution to the protection against nitric oxide remains unclear.
The previous search for macrophage-induced genes of animal C. pseudotuberculosis by means of a cloned promoter library provided two gene tags showing significant induction rates in macrophages [4]. The nucleotide sequence of the respective gene tags revealed similarity to nonribosomal peptide synthetases (44-fold induction) and to the β-subunit of acyl-CoA carboxylases (24-fold induction), respectively. The genome sequence of C. pseudotuberculosis FRC41 encodes two nonribosomal peptide synthetases, NrpS1 and NrpS2 (Table 1). These modular enzymes are used by microorganisms to participate in the synthesis of many secondary metabolites, including for instance siderophores and antibiotics [93]. As both nrpS genes were not assigned to the iron-responsive DtxR regulon of C. pseudotuberculosis FRC41 and siderophore biosynthesis is carried out by alternative pathways independent of nonribosomal peptide synthetases, a physiological role in iron metabolism of the two proteins cannot be deduced from the current data. However, the strong upregulation of gene expression in macrophages points toward a protective or toxic function during the infection of at least one nonribosomal peptide synthetase [4]. A role in virulence of a secondary metabolite produced by a nonribosomal peptide synthetase has been demonstrated in Streptomyces acidiscabies. This phytopathogen produces thaxtomin A which is necessary for the infection of potato tubers [94].
Three genes coding for β-subunits of acyl-CoA carboxylases are present in the genome of C. pseudotuberculosis FRC41 (Table 1). These genes are located in highly conserved regions of corynebacterial genomes and are essential for either fatty acid synthesis (dtsR1) or mycolic acid synthesis (dtsR2 and accD3) [95]. The acetyl-CoA carboxylase of C. glutamicum consists of the biotinylated α-subunit AccBC, the β-subunit AccD1 (DtsR1) and the small AccE protein. The acyl-CoA carboxylase involved in mycolic acid synthesis of C. glutamicum consists of the two β-subunits AccD2 and AccD3 (DtsR2 and AccD3) in addition to AccBC and AccE [95, 96]. The expression of the respective genes in C. pseudotuberculosis FRC41 is probably controlled by the global transcription regulator GlxR (Figure 6), providing hints that this regulatory protein has a key function in connecting both the central metabolism and the expression of candidate virulence factors in this pathogenic bacterium. Mycolic acids, which are long-chain α-alkyl-β-hydroxy fatty acids, are important components of the corynebacterial cell wall [97] and probably associated with the pathogenicity of C. pseudotuberculosis[98]. They can provide a thick layer at the outer surface of the cell that protects the bacterium from antibiotics and the host's immune system [99]. Moreover, the noncovalently bound trehalose dimycolate is a well-established immunostimulatory compound with toxic properties [97]. Variations in the amount of mycolic acids and differences in other cell surface properties provide a basis for explaining the aggregation capacity of C. pseudotuberculosis in suspension, the strong aggregation within macrophages, the formation of thick capsules after the release of the pathogen from macrophages [11], and the occurrence of virulent and attenuated strains [98].
The complete genome sequence of C. pseudotuberculosis FRC41 of human origin provides detailed insights into the gene repertoire contributing to the virulence of this bacterium that was isolated from a rare case of necrotizing lymphadenitis [12]. By combining in silico data obtained from the genome annotation with previous experimental knowledge, occasional observations on genes that affect the virulence of C. pseudotuberculosis were integrated into a global view on the pathogenicity of this species. The reconstruction of the DtxR regulon for instance provides a comprehensive set of genes involved in the acquisition of iron and extends the initial observation that a fagB mutant was not impaired in iron uptake [29]. A systematic mutational characterization of the newly detected iron transporters and siderophore biosynthesis gene clusters may help to dissect the contribution of each system to the virulence of C. pseudotuberculosis FRC41. It is very likely that the ciu siderophore biosynthesis and transport system may complement the uptake of iron by the FagABCD transporter under certain environmental conditions, as the ciuA gene was shown earlier to be expressed in C. pseudotuberculosis[1]. Likewise, a reporter transposon system indicated that a gene coding for a fimbrial subunit is expressed during growth of C. pseudotuberculosis in standard medium [40]. As the genome sequence of C. pseudotuberculosis FRC41 revealed two gene clusters encoding adhesive pili, it is interesting to examine how the respective pilin monomers or pilus polymers contribute to the adherence of C. pseudotuberculosis to host tissue and how their expression is controlled. We detected a DNA-binding site for the cAMP-sensing transcription regulator GlxR in the spaA-srtB intergenic region, whereas the iron-responsive regulator DtxR was proposed to control the assembly of pilin subunits in C. diphtheriae NCTC 13129 [100]. Another previous study provided two DNA sequence tags for macrophage-induced genes [4] that were linked to the nonribosomal peptide synthetase genes nrpS1 and nrpS2 and to the acyl-CoA carboxylase subunit genes dtsR1, dtsR2 and accD3 by means of the genome sequence of C. pseudotuberculosis FRC41. The detection of the latter genes is consistent with the observation that the cell surface of C. pseudotuberculosis is an important factor contributing to virulence [98]. Future work should compare the knowledge deduced from the genome sequence of C. pseudotuberculosis FRC41 with the genetic information generated from other human isolates or animal pathogens of the different biovars [101], or even extend the genome comparison to the pangenomic level. This global comparative approach with a larger set of sequenced genomes may provide comprehensive insights into the distinctive features of each biovar or strain. As the currently available commercial vaccines are unable to fully protect susceptible animals [102, 103], the extended knowledge of potential virulence factors and novel antigens of C. pseudotuberculosis might be helpful for the design of more effective vaccines and molecular diagnostics to control caseous lymphadenitis in sheep and goats to reduce thereby the occupational risk exposure for humans.
Bacterial strain and growth conditions
C. pseudotuberculosis FRC41 was isolated from the inguinal lymph node of a 12-year-old French girl with necrotizing lymphadenitis [12]. The taxonomic identification of this clinical isolate was recently confirmed using multiplex PCR and the nucleotide sequence of the pld gene (N. Guiso, unpublished data). C. pseudotuberculosis FRC41 was routinely grown in brain-heart-infusion (BHI) broth or on Columbia agar with sheep blood at 37°C.
Preparation of chromosomal DNA for genome sequencing
The preparation of chromosomal DNA from C. pseudotuberculosis FRC41 was performed as follows: 50-ml aliquots of cultures grown for 48-72 h were centrifuged at 4°C and 2,000 × g for 20 min. The cell pellets were resuspended in 0.6 ml Tris/NaCl buffer [10 mM Tris (pH 7.0), 10 mM EDTA (pH 8.0), 300 mM NaCl] and transferred to VK01 Precellys lysing tubes. The cells were lysed by means of a Precellys 24-Dual Tissue Homogenizer, using two cycles of 6,500 rpm for 15 sec with an interval of 30 sec. The chromosomal DNA was purified by phenol/chloroform/isoamyl alcohol (25:24:1) extraction and precipitated with ethanol. The DNA concentration was determined with a Tecan Infinite 200 Microplate Reader.
Sequencing of the C. pseudotuberculosis FRC41 genome
A total of 5 μg of purified chromosomal DNA was used for constructing a single-stranded template DNA library. The DNA concentration of the library was measured by using the Agilent RNA 6000 Nano Kit. The preparation of the single-stranded template DNA library and DNA sequencing were performed according to manufacturer's protocols (Roche Applied Science). The sequencing of C. pseudotuberculosis DNA was carried out with the Genome Sequencer FLX Instrument and Titanium chemistry (Roche Applied Science). The sequence data were assembled with the GS de novo Assembler Software (Version 2.3). According to the 454 Newbler Metrics file, 286,938 reads representing 94,447,635 bases were assembled. Using the default cutoff of 500 bases for the size classification of the contigs, ten large contigs (≥ 500 bases) and one small contig (313 bases) were obtained to give a total size of 2,319,243 bases. The gap closure process was supported by the related reference contig arrangement tool r2cat, using the C. diphtheriae NCTC 13129 genome sequence as a reference [13]. The remaining gaps in the genome sequence were closed by PCR with Phusion hot start high-fidelity DNA polymerase (Finnzymes) and genomic template DNA. All primers used in this study were synthesized by Metabion. The PCR assays were carried out with a TProfessional PCR thermocycler (Biometra) according to standard protocols (Finnzymes). The amplified DNA fragments linking the individual contigs were sequenced by IIT Biotech. All DNA sequences were uploaded into the Consed program [104] to generate the complete genome sequence of C. pseudotuberculosis FRC41.
Bioinformatic analysis of the complete genome sequence
The assembled sequence of C. pseudotuberculosis FRC41 was uploaded into the bacterial genome annotation system GenDB [15]. The annotation of the complete genome sequence was performed as described previously [105], followed by manual curation. Analyses of the predicted gene content and the metabolic properties of C. pseudotuberculosis FRC41 were accomplished by the computer programs EDGAR [19] and CARMEN [106]. The synteny between the genomes of C. pseudotuberculosis FRC41 and C. diphtheriae NCTC 13129 was calculated by the EDGAR software [19]. The origin of chromosomal DNA replication was predicted with the Ori-Finder tool [17]. The genome sequence of C. pseudotuberculosis FRC41 has been deposited in the GenBank database with accession number CP002097.
Bioinformatic analysis of the regulatory repertoire
The detection of the transcriptional regulatory repertoire of C. pseudotuberculosis FRC41 was performed by a combined bioinformatic approach using several tools and programs [26]. Proteins containing putative DNA-binding domains were detected by means of the HMM library and genome assignments server Superfamily version 1.75 [107]. To identify among the set of potential DNA-binding proteins those representing transcription regulators, hidden Markov model (HMM) profiles of regulatory protein families were downloaded from the Pfam database version 24.0 [108] and used for searches against the predicted C. pseudotuberculosis FRC41 proteins by applying the HMMsearch module of the profile hidden Markov model software HMMER [109]. Moreover, the helix-turn-helix (HTH) recognition tool [110] integrated in the GenDB platform was applied to scan the putative DNA-binding transcription regulators for the presence of HTH motifs. The classification of C. pseudotuberculosis FRC41 proteins into Clusters of Orthologous Groups of proteins [111] during genome annotation provided further indications on the role of the predicted proteins in transcriptional regulation. The genome-wide search was extended by using data on known transcription regulators from C. glutamicum[23]. A validation step was included by performing BLASTP [112] searches against the NCBI protein database and evaluating results generated with the Conserved Domain Search program [56]. During the final step of data analysis, the DNA-binding transcription regulators of C. pseudotuberculosis FRC41 were grouped into regulatory protein families [23, 26].
The detection of DNA-binding sites in the genome sequence of C. pseudotuberculosis FRC41 followed a combined workflow, using both position weight matrices and hidden Markov models [74]. The programs PoSSuMsearch [113] and HMMsearch [109] were applied to scan the complete genome sequence of C. pseudotuberculosis FRC41. As HMMsearch does not support searching in both directions of a double strand, scanning of the reverse complementary DNA sequence was implemented. PoSSuMsearch was configured for lazy probability evaluation [113]. The comprehensive set of validated DNA-binding sites sequences from C. glutamicum was downloaded from the reference database CoryneRegNet [114] and used as input for PoSSuMsearch and HMMsearch. The respective gene-regulatory network transfer between C. glutamicum and other corynebacteria on the genome-scale was described previously [114]. E-value cut-offs were used as described for the genome-wide pattern recognition approach in C. glutamicum[75]. Sequence logos of the detected DNA-binding sites were generated with WebLogo version 2.8.2 [115].
ET and JSchn acknowledge the receipt of scholarships which are embedded into the Cluster Industrial Biotechnology CLIB2021 and financed by Bielefeld University and the Ministry of Innovation, Science, Research and Technology of North Rhine-Westphalia. LO and AB were supported by the Deutsche Forschungsgemeinschaft in frame of SFB 796 (project B5). The valuable contribution of the CeBiTec sequencing team (A. Albersmeier, K. Brinkrolf, S. Schatschneider, R. Szczepanowski, P. Viehoever, A. Winkler) to this genome project is gratefullly acknowledged. The authors thank the sequencing team from IIT Biotech for the help during the gap closure process.
Authors' Affiliations
- Dorella FA, Pacheco LG, Oliveira SC, Miyoshi A, Azevedo V: Corynebacterium pseudotuberculosis: microbiology, biochemical properties, pathogenesis and molecular studies of virulence. Vet Res. 2006, 37: 201-218. 10.1051/vetres:2005056.PubMedGoogle Scholar
- Baird GJ, Fontaine MC: Corynebacterium pseudotuberculosis and its role in ovine caseous lymphadenitis. J Comp Pathol. 2007, 137: 179-210. 10.1016/j.jcpa.2007.07.002.PubMedGoogle Scholar
- Paton MW, Rose IR, Hart RA, Sutherland SS, Mercy AR, Ellis TM, Dhaliwal JA: New infection with Corynebacterium pseudotuberculosis reduces wool production. Aust Vet J. 1994, 71: 47-49. 10.1111/j.1751-0813.1994.tb06152.x.PubMedGoogle Scholar
- McKean S, Davies J, Moore R: Identification of macrophage induced genes of Corynebacterium pseudotuberculosis by differential fluorescence induction. Microbes Infect. 2005, 7: 1352-1363. 10.1016/j.micinf.2005.05.002.PubMedGoogle Scholar
- McNamara PJ, Bradley GA, Songer JG: Targeted mutagenesis of the phospholipase D gene results in decreased virulence of Corynebacterium pseudotuberculosis. Mol Microbiol. 1994, 12: 921-930. 10.1111/j.1365-2958.1994.tb01080.x.PubMedGoogle Scholar
- Songer JG: Bacterial phospholipases and their role in virulence. Trends Microbiol. 1997, 5: 156-161. 10.1016/S0966-842X(97)01005-6.PubMedGoogle Scholar
- Bregenzer T, Frei R, Ohnacker H, Zimmerli W: Corynebacterium pseudotuberculosis infection in a butcher. Clin Microbiol Infect. 1997, 3: 696-698. 10.1111/j.1469-0691.1997.tb00482.x.PubMedGoogle Scholar
- Peel MM, Palmer GG, Stacpoole AM, Kerr TG: Human lymphadenitis due to Corynebacterium pseudotuberculosis: report of ten cases from Australia and review. Clin Infect Dis. 1997, 24: 185-191.PubMedGoogle Scholar
- Funke G, von Graevenitz A, Clarridge JE, Bernard KA: Clinical microbiology of coryneform bacteria. Clin Microbiol Rev. 1997, 10: 125-159.PubMed CentralPubMedGoogle Scholar
- Keslin MH, McCoy EL, McCusker JJ, Lutch JS: Corynebacterium pseudotuberculosis. A new cause of infectious and eosinophilic pneumonia. Am J Med. 1979, 67: 228-231. 10.1016/0002-9343(79)90395-4.PubMedGoogle Scholar
- Walker J, Jackson HJ, Eggleton DG, Meeusen EN, Wilson MJ, Brandon MR: Identification of a novel antigen from Corynebacterium pseudotuberculosis that protects sheep against caseous lymphadenitis. Infect Immun. 1994, 62: 2562-2567.PubMed CentralPubMedGoogle Scholar
- Join-Lambert OF, Ouache M, Canioni D, Beretti JL, Blanche S, Berche P, Kayal S: Corynebacterium pseudotuberculosis necrotizing lymphadenitis in a twelve-year-old patient. Pediatr Infect Dis J. 2006, 25: 848-851. 10.1097/01.inf.0000234071.93044.77.PubMedGoogle Scholar
- Husemann P, Stoye J: r2cat: synteny plots and comparative assembly. Bioinformatics. 2010, 26: 570-571. 10.1093/bioinformatics/btp690.PubMed CentralPubMedGoogle Scholar
- Cerdeno-Tarraga AM, Efstratiou A, Dover LG, Holden MT, Pallen M, Bentley SD, Besra GS, Churcher C, James KD, De Zoysa A, et al: The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129. Nucleic Acids Res. 2003, 31: 6516-6523. 10.1093/nar/gkg874.PubMed CentralPubMedGoogle Scholar
- Meyer F, Goesmann A, McHardy AC, Bartels D, Bekel T, Clausen J, Kalinowski J, Linke B, Rupp O, Giegerich R, Pühler A: GenDB-an open source genome annotation system for prokaryote genomes. Nucleic Acids Res. 2003, 31: 2187-2195. 10.1093/nar/gkg312.PubMed CentralPubMedGoogle Scholar
- Lowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997, 25: 955-964. 10.1093/nar/25.5.955.PubMed CentralPubMedGoogle Scholar
- Gao F, Zhang CT: Ori-Finder: a web-based system for finding oriCs in unannotated bacterial genomes. BMC Bioinformatics. 2008, 9: 79-10.1186/1471-2105-9-79.PubMed CentralPubMedGoogle Scholar
- Hendrickson H, Lawrence JG: Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites. Mol Microbiol. 2007, 64: 42-56. 10.1111/j.1365-2958.2007.05596.x.PubMedGoogle Scholar
- Blom J, Albaum SP, Doppmeier D, Pühler A, Vorhölter FJ, Zakrzewski M, Goesmann A: EDGAR: a software framework for the comparative analysis of prokaryotic genomes. BMC Bioinformatics. 2009, 10: 154-10.1186/1471-2105-10-154.PubMed CentralPubMedGoogle Scholar
- Ventura M, Canchaya C, Tauch A, Chandra G, Fitzgerald GF, Chater KF, van Sinderen D: Genomics of Actinobacteria: tracing the evolutionary history of an ancient phylum. Microbiol Mol Biol Rev. 2007, 71: 495-548. 10.1128/MMBR.00005-07.PubMed CentralPubMedGoogle Scholar
- Trost E, Götker S, Schneider J, Schneiker-Bekel S, Szczepanowski R, Tilker A, Viehoever P, Arnold W, Bekel T, Blom J, et al: Complete genome sequence and lifestyle of black-pigmented Corynebacterium aurimucosum ATCC 700975 (formerly C. nigricans CN-1) isolated from a vaginal swab of a woman with spontaneous abortion. BMC Genomics. 2010, 11: 91-10.1186/1471-2164-11-91.PubMed CentralPubMedGoogle Scholar
- Yozwiak ML, Songer JG: Effect of Corynebacterium pseudotuberculosis phospholipase D on viability and chemotactic responses of ovine neutrophils. Am J Vet Res. 1993, 54: 392-397.PubMedGoogle Scholar
- Schröder J, Tauch A: Transcriptional regulation of gene expression in Corynebacterium glutamicum: the role of global, master and local regulators in the modular and hierarchical gene regulatory network. FEMS Microbiol Rev. 2010, 34: 685-737.PubMedGoogle Scholar
- Rodionov DA: Comparative genomic reconstruction of transcriptional regulatory networks in bacteria. Chem Rev. 2007, 107: 3467-3497. 10.1021/cr068309+.PubMed CentralPubMedGoogle Scholar
- Brinkrolf K, Brune I, Tauch A: The transcriptional regulatory network of the amino acid producer Corynebacterium glutamicum. J Biotechnol. 2007, 129: 191-211. 10.1016/j.jbiotec.2006.12.013.PubMedGoogle Scholar
- Brune I, Brinkrolf K, Kalinowski J, Pühler A, Tauch A: The individual and common repertoire of DNA-binding transcriptional regulators of Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium diphtheriae and Corynebacterium jeikeium deduced from the complete genome sequences. BMC Genomics. 2005, 6: 86-10.1186/1471-2164-6-86.PubMed CentralPubMedGoogle Scholar
- Ramos JL, Martinez-Bueno M, Molina-Henares AJ, Teran W, Watanabe K, Zhang X, Gallegos MT, Brennan R, Tobes R: The TetR family of transcriptional repressors. Microbiol Mol Biol Rev. 2005, 69: 326-356. 10.1128/MMBR.69.2.326-356.2005.PubMed CentralPubMedGoogle Scholar
- Andrews SC, Robinson AK, Rodriguez-Quinones F: Bacterial iron homeostasis. FEMS Microbiol Rev. 2003, 27: 215-237. 10.1016/S0168-6445(03)00055-X.PubMedGoogle Scholar
- Billington SJ, Esmay PA, Songer JG, Jost BH: Identification and role in virulence of putative iron acquisition genes from Corynebacterium pseudotuberculosis. FEMS Microbiol Lett. 2002, 208: 41-45. 10.1111/j.1574-6968.2002.tb11058.x.PubMedGoogle Scholar
- Oram DM, Avdalovic A, Holmes RK: Analysis of genes that encode DtxR-like transcriptional regulators in pathogenic and saprophytic corynebacterial species. Infect Immun. 2004, 72: 1885-1895. 10.1128/IAI.72.4.1885-1895.2004.PubMed CentralPubMedGoogle Scholar
- Brune I, Werner H, Hüser AT, Kalinowski J, Pühler A, Tauch A: The DtxR protein acting as dual transcriptional regulator directs a global regulatory network involved in iron metabolism of Corynebacterium glutamicum. BMC Genomics. 2006, 7: 21-10.1186/1471-2164-7-21.PubMed CentralPubMedGoogle Scholar
- Drazek ES, Hammack CA, Schmitt MP: Corynebacterium diphtheriae genes required for acquisition of iron from haemin and haemoglobin are homologous to ABC haemin transporters. Mol Microbiol. 2000, 36: 68-84. 10.1046/j.1365-2958.2000.01818.x.PubMedGoogle Scholar
- Allen CE, Schmitt MP: HtaA is an iron-regulated hemin binding protein involved in the utilization of heme iron in Corynebacterium diphtheriae. J Bacteriol. 2009, 191: 2638-2648. 10.1128/JB.01784-08.PubMed CentralPubMedGoogle Scholar
- Schmitt MP: Utilization of host iron sources by Corynebacterium diphtheriae: identification of a gene whose product is homologous to eukaryotic heme oxygenases and is required for acquisition of iron from heme and hemoglobin. J Bacteriol. 1997, 179: 838-845.PubMed CentralPubMedGoogle Scholar
- Schmitt MP: Transcription of the Corynebacterium diphtheriae hmuO gene is regulated by iron and heme. Infect Immun. 1997, 65: 4634-4641.PubMed CentralPubMedGoogle Scholar
- Bibb LA, Kunkle CA, Schmitt MP: The ChrA-ChrS and HrrA-HrrS signal transduction systems are required for activation of the hmuO promoter and repression of the hemA promoter in Corynebacterium diphtheriae. Infect Immun. 2007, 75: 2421-2431. 10.1128/IAI.01821-06.PubMed CentralPubMedGoogle Scholar
- Crosa JH, Walsh CT: Genetics and assembly line enzymology of siderophore biosynthesis in bacteria. Microbiol Mol Biol Rev. 2002, 66: 223-249. 10.1128/MMBR.66.2.223-249.2002.PubMed CentralPubMedGoogle Scholar
- Miethke M, Marahiel MA: Siderophore-based iron acquisition and pathogen control. Microbiol Mol Biol Rev. 2007, 71: 413-451. 10.1128/MMBR.00012-07.PubMed CentralPubMedGoogle Scholar
- Challis GL: A widely distributed bacterial pathway for siderophore biosynthesis independent of nonribosomal peptide synthetases. Chembiochem. 2005, 6: 601-611. 10.1002/cbic.200400283.PubMedGoogle Scholar
- Dorella FA, Estevam EM, Pacheco LG, Guimaraes CT, Lana UG, Gomes EA, Barsante MM, Oliveira SC, Meyer R, Miyoshi A, Azevedo V: In vivo insertional mutagenesis in Corynebacterium pseudotuberculosis: an efficient means to identify DNA sequences encoding exported proteins. Appl Environ Microbiol. 2006, 72: 7368-7372. 10.1128/AEM.00294-06.PubMed CentralPubMedGoogle Scholar
- Kunkle CA, Schmitt MP: Analysis of a DtxR-regulated iron transport and siderophore biosynthesis gene cluster in Corynebacterium diphtheriae. J Bacteriol. 2005, 187: 422-433. 10.1128/JB.187.2.422-433.2005.PubMed CentralPubMedGoogle Scholar
- Wennerhold J, Krug A, Bott M: The AraC-type regulator RipA represses aconitase and other iron proteins from Corynebacterium under iron limitation and is itself repressed by DtxR. J Biol Chem. 2005, 280: 40500-40508. 10.1074/jbc.M508693200.PubMedGoogle Scholar
- Kana BD, Weinstein EA, Avarbock D, Dawes SS, Rubin H, Mizrahi V: Characterization of the cydAB-encoded cytochrome bd oxidase from Mycobacterium smegmatis. J Bacteriol. 2001, 183: 7076-7086. 10.1128/JB.183.24.7076-7086.2001.PubMed CentralPubMedGoogle Scholar
- Martinez A, Kolter R: Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps. J Bacteriol. 1997, 179: 5188-5194.PubMed CentralPubMedGoogle Scholar
- Zhao G, Ceci P, Ilari A, Giangiacomo L, Laue TM, Chiancone E, Chasteen ND: Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli. J Biol Chem. 2002, 277: 27689-27696. 10.1074/jbc.M202094200.PubMedGoogle Scholar
- Gordon BR, Li Y, Wang L, Sintsova A, van Bakel H, Tian S, Navarre WW, Xia B, Liu J: Lsr2 is a nucleoid-associated protein that targets AT-rich sequences and virulence genes in Mycobacterium tuberculosis. Proc Natl Acad Sci USA. 2010, 107: 5154-5159. 10.1073/pnas.0913551107.PubMed CentralPubMedGoogle Scholar
- Colangeli R, Haq A, Arcus VL, Summers E, Magliozzo RS, McBride A, Mitra AK, Radjainia M, Khajo A, Jacobs WR, et al: The multifunctional histone-like protein Lsr2 protects mycobacteria against reactive oxygen intermediates. Proc Natl Acad Sci USA. 2009, 106: 4414-4418. 10.1073/pnas.0810126106.PubMed CentralPubMedGoogle Scholar
- Zaika EI, Perlow RA, Matz E, Broyde S, Gilboa R, Grollman AP, Zharkov DO: Substrate discrimination by formamidopyrimidine-DNA glycosylase: a mutational analysis. J Biol Chem. 2004, 279: 4849-4861. 10.1074/jbc.M310262200.PubMedGoogle Scholar
- Hassett DJ, Cohen MS: Bacterial adaptation to oxidative stress: implications for pathogenesis and interaction with phagocytic cells. Faseb J. 1989, 3: 2574-2582.PubMedGoogle Scholar
- Nathan C, Shiloh MU: Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci USA. 2000, 97: 8841-8848. 10.1073/pnas.97.16.8841.PubMed CentralPubMedGoogle Scholar
- Zahrt TC, Deretic V: Reactive nitrogen and oxygen intermediates and bacterial defenses: unusual adaptations in Mycobacterium tuberculosis. Antioxid Redox Signal. 2002, 4: 141-159. 10.1089/152308602753625924.PubMedGoogle Scholar
- Storz G, Tartaglia LA: OxyR: a regulator of antioxidant genes. J Nutr. 1992, 122: 627-630.PubMedGoogle Scholar
- Wood ZA, Schroder E, Robin Harris J, Poole LB: Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci. 2003, 28: 32-40. 10.1016/S0968-0004(02)00003-8.PubMedGoogle Scholar
- Guimaraes BG, Souchon H, Honore N, Saint-Joanis B, Brosch R, Shepard W, Cole ST, Alzari PM: Structure and mechanism of the alkyl hydroperoxidase AhpC, a key element of the Mycobacterium tuberculosis defense system against oxidative stress. J Biol Chem. 2005, 280: 25735-25742. 10.1074/jbc.M503076200.PubMedGoogle Scholar
- Bryk R, Griffin P, Nathan C: Peroxynitrite reductase activity of bacterial peroxiredoxins. Nature. 2000, 407: 211-215. 10.1038/35025109.PubMedGoogle Scholar
- Marchler-Bauer A, Anderson JB, Derbyshire MK, DeWeese-Scott C, Gonzales NR, Gwadz M, Hao L, He S, Hurwitz DI, Jackson JD, et al: CDD: a conserved domain database for interactive domain family analysis. Nucleic Acids Res. 2007, 35: D237-240. 10.1093/nar/gkl951.PubMed CentralPubMedGoogle Scholar
- Merkamm M, Guyonvarch A: Cloning of the sodA gene from Corynebacterium melassecola and role of superoxide dismutase in cellular viability. J Bacteriol. 2001, 183: 1284-1295. 10.1128/JB.2001.183.4.1284-1295.2001.PubMed CentralPubMedGoogle Scholar
- Babu MM, Priya ML, Selvan AT, Madera M, Gough J, Aravind L, Sankaran K: A database of bacterial lipoproteins (DOLOP) with functional assignments to predicted lipoproteins. J Bacteriol. 2006, 188: 2761-2773. 10.1128/JB.188.8.2761-2773.2006.PubMed CentralPubMedGoogle Scholar
- Dussurget O, Stewart G, Neyrolles O, Pescher P, Young D, Marchal G: Role of Mycobacterium tuberculosis copper-zinc superoxide dismutase. Infect Immun. 2001, 69: 529-533. 10.1128/IAI.69.1.529-533.2001.PubMed CentralPubMedGoogle Scholar
- Piddington DL, Fang FC, Laessig T, Cooper AM, Orme IM, Buchmeier NA: Cu,Zn superoxide dismutase of Mycobacterium tuberculosis contributes to survival in activated macrophages that are generating an oxidative burst. Infect Immun. 2001, 69: 4980-4987. 10.1128/IAI.69.8.4980-4987.2001.PubMed CentralPubMedGoogle Scholar
- Wilks KE, Dunn KL, Farrant JL, Reddin KM, Gorringe AR, Langford PR, Kroll JS: Periplasmic superoxide dismutase in meningococcal pathogenicity. Infect Immun. 1998, 66: 213-217.PubMed CentralPubMedGoogle Scholar
- San Mateo LR, Toffer KL, Orndorff PE, Kawula TH: Neutropenia restores virulence to an attenuated Cu,Zn superoxide dismutase-deficient Haemophilus ducreyi strain in the swine model of chancroid. Infect Immun. 1999, 67: 5345-5351.PubMed CentralPubMedGoogle Scholar
- Papp-Wallace KM, Maguire ME: Manganese transport and the role of manganese in virulence. Annu Rev Microbiol. 2006, 60: 187-209. 10.1146/annurev.micro.60.080805.142149.PubMedGoogle Scholar
- Schmitt MP: Analysis of a DtxR-like metalloregulatory protein, MntR, from Corynebacterium diphtheriae that controls expression of an ABC metal transporter by an Mn(2+)-dependent mechanism. J Bacteriol. 2002, 184: 6882-6892. 10.1128/JB.184.24.6882-6892.2002.PubMed CentralPubMedGoogle Scholar
- Lee JW, Helmann JD: Functional specialization within the Fur family of metalloregulators. Biometals. 2007, 20: 485-499. 10.1007/s10534-006-9070-7.PubMedGoogle Scholar
- Schröder J, Jochmann N, Rodionov DA, Tauch A: The Zur regulon of Corynebacterium glutamicum ATCC 13032. BMC Genomics. 2010, 11: 12-PubMed CentralPubMedGoogle Scholar
- Grover A, Sharma R: Identification and characterization of a major Zn(II) resistance determinant of Mycobacterium smegmatis. J Bacteriol. 2006, 188: 7026-7032. 10.1128/JB.00643-06.PubMed CentralPubMedGoogle Scholar
- Riccardi G, Milano A, Pasca MR, Nies DH: Genomic analysis of zinc homeostasis in Mycobacterium tuberculosis. FEMS Microbiol Lett. 2008, 287: 1-7. 10.1111/j.1574-6968.2008.01320.x.PubMedGoogle Scholar
- Haas CE, Rodionov DA, Kropat J, Malasarn D, Merchant SS, de Crecy-Lagard V: A subset of the diverse COG0523 family of putative metal chaperones is linked to zinc homeostasis in all kingdoms of life. BMC Genomics. 2009, 10: 470-10.1186/1471-2164-10-470.PubMed CentralPubMedGoogle Scholar
- Navarre WW, Schneewind O: Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol Mol Biol Rev. 1999, 63: 174-229.PubMed CentralPubMedGoogle Scholar
- Ton-That H, Schneewind O: Assembly of pili in Gram-positive bacteria. Trends Microbiol. 2004, 12: 228-234. 10.1016/j.tim.2004.03.004.PubMedGoogle Scholar
- Ton-That H, Marraffini LA, Schneewind O: Sortases and pilin elements involved in pilus assembly of Corynebacterium diphtheriae. Mol Microbiol. 2004, 53: 251-261. 10.1111/j.1365-2958.2004.04117.x.PubMedGoogle Scholar
- Swaminathan A, Mandlik A, Swierczynski A, Gaspar A, Das A, Ton-That H: Housekeeping sortase facilitates the cell wall anchoring of pilus polymers in Corynebacterium diphtheriae. Mol Microbiol. 2007, 66: 961-974. 10.1111/j.1365-2958.2007.05968.x.PubMed CentralPubMedGoogle Scholar
- Kohl TA, Baumbach J, Jungwirth B, Pühler A, Tauch A: The GlxR regulon of the amino acid producer Corynebacterium glutamicum in silico and in vitro detection of DNA binding sites of a global transcription regulator. J Biotechnol. 2008, 135: 340-350. 10.1016/j.jbiotec.2008.05.011.PubMedGoogle Scholar
- Kohl TA, Tauch A: The GlxR regulon of the amino acid producer Corynebacterium glutamicum: Detection of the corynebacterial core regulon and integration into the transcriptional regulatory network model. J Biotechnol. 2009, 143: 239-246. 10.1016/j.jbiotec.2009.08.005.PubMedGoogle Scholar
- Mandlik A, Das A, Ton-That H: The molecular switch that activates the cell wall anchoring step of pilus assembly in gram-positive bacteria. Proc Natl Acad Sci USA. 2008, 105: 14147-14152. 10.1073/pnas.0806350105.PubMed CentralPubMedGoogle Scholar
- Mandlik A, Swierczynski A, Das A, Ton-That H: Corynebacterium diphtheriae employs specific minor pilins to target human pharyngeal epithelial cells. Mol Microbiol. 2007, 64: 111-124. 10.1111/j.1365-2958.2007.05630.x.PubMed CentralPubMedGoogle Scholar
- McKean SC, Davies JK, Moore RJ: Probing the heat shock response of Corynebacterium pseudotuberculosis: the major virulence factor, phospholipase D, is downregulated at 43 degrees C. Res Microbiol. 2007, 158: 279-286. 10.1016/j.resmic.2006.12.006.PubMedGoogle Scholar
- McKean SC, Davies JK, Moore RJ: Expression of phospholipase D, the major virulence factor of Corynebacterium pseudotuberculosis, is regulated by multiple environmental factors and plays a role in macrophage death. Microbiology. 2007, 153: 2203-2211. 10.1099/mic.0.2007/005926-0.PubMedGoogle Scholar
- Wilson MJ, Brandon MR, Walker J: Molecular and biochemical characterization of a protective 40-kilodalton antigen from Corynebacterium pseudotuberculosis. Infect Immun. 1995, 63: 206-211.PubMed CentralPubMedGoogle Scholar
- Dubin G: Extracellular proteases of Staphylococcus spp. Biol Chem. 2002, 383: 1075-1086. 10.1515/BC.2002.116.PubMedGoogle Scholar
- Mattos-Guaraldi AL, Duarte Formiga LC, Pereira GA: Cell surface components and adhesion in Corynebacterium diphtheriae. Microbes Infect. 2000, 2: 1507-1512. 10.1016/S1286-4579(00)01305-8.PubMedGoogle Scholar
- Vimr ER, Kalivoda KA, Deszo EL, Steenbergen SM: Diversity of microbial sialic acid metabolism. Microbiol Mol Biol Rev. 2004, 68: 132-153. 10.1128/MMBR.68.1.132-153.2004.PubMed CentralPubMedGoogle Scholar
- Kim S, Oh DB, Kwon O, Kang HA: Identification and functional characterization of the NanH extracellular sialidase from Corynebacterium diphtheriae. J Biochem. 2010, 147: 523-533. 10.1093/jb/mvp198.PubMedGoogle Scholar
- Moreira Lde O, Andrade AF, Vale MD, Souza SM, Hirata R, Asad LM, Asad NR, Monteiro-Leal LH, Previato JO, Mattos-Guaraldi AL: Effects of iron limitation on adherence and cell surface carbohydrates of Corynebacterium diphtheriae strains. Appl Environ Microbiol. 2003, 69: 5907-5913. 10.1128/AEM.69.10.5907-5913.2003.Google Scholar
- Ott L, Holler M, Gerlach RG, Hensel M, Rheinlaender J, Schaffer TE, Burkovski A: Corynebacterium diphtheriae invasion-associated protein (DIP1281) is involved in cell surface organization, adhesion and internalization in epithelial cells. BMC Microbiol. 2010, 10: 2-10.1186/1471-2180-10-2.PubMed CentralPubMedGoogle Scholar
- Kana BD, Mizrahi V: Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling. FEMS Immunol Med Microbiol. 2010, 58: 39-50. 10.1111/j.1574-695X.2009.00606.x.PubMedGoogle Scholar
- Boneca IG: The role of peptidoglycan in pathogenesis. Curr Opin Microbiol. 2005, 8: 46-53. 10.1016/j.mib.2004.12.008.PubMedGoogle Scholar
- Lenz LL, Mohammadi S, Geissler A, Portnoy DA: SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis. Proc Natl Acad Sci USA. 2003, 100: 12432-12437. 10.1073/pnas.2133653100.PubMed CentralPubMedGoogle Scholar
- Jungwirth B, Emer D, Brune I, Hansmeier N, Pühler A, Eikmanns BJ, Tauch A: Triple transcriptional control of the resuscitation promoting factor 2 (rpf2) gene of Corynebacterium glutamicum by the regulators of acetate metabolism RamA and RamB and the cAMP-dependent regulator GlxR. FEMS Microbiol Lett. 2008, 281: 190-197. 10.1111/j.1574-6968.2008.01098.x.PubMedGoogle Scholar
- Luthra A, Malik SS, Ramachandran R: Cloning, purification and comparative structural analysis of two hypothetical proteins from Mycobacterium tuberculosis found in the human granuloma during persistence and highly up-regulated under carbon-starvation conditions. Protein Expr Purif. 2008, 62: 64-74. 10.1016/j.pep.2008.06.011.PubMedGoogle Scholar
- Tucker NP, D'Autreaux B, Studholme DJ, Spiro S, Dixon R: DNA binding activity of the Escherichia coli nitric oxide sensor NorR suggests a conserved target sequence in diverse proteobacteria. J Bacteriol. 2004, 186: 6656-6660. 10.1128/JB.186.19.6656-6660.2004.PubMed CentralPubMedGoogle Scholar
- Donadio S, Monciardini P, Sosio M: Polyketide synthases and nonribosomal peptide synthetases: the emerging view from bacterial genomics. Nat Prod Rep. 2007, 24: 1073-1109. 10.1039/b514050c.PubMedGoogle Scholar
- Healy FG, Wach M, Krasnoff SB, Gibson DM, Loria R: The txtAB genes of the plant pathogen Streptomyces acidiscabies encode a peptide synthetase required for phytotoxin thaxtomin A production and pathogenicity. Mol Microbiol. 2000, 38: 794-804. 10.1046/j.1365-2958.2000.02170.x.PubMedGoogle Scholar
- Gande R, Dover LG, Krumbach K, Besra GS, Sahm H, Oikawa T, Eggeling L: The two carboxylases of Corynebacterium glutamicum essential for fatty acid and mycolic acid synthesis. J Bacteriol. 2007, 189: 5257-5264. 10.1128/JB.00254-07.PubMed CentralPubMedGoogle Scholar
- Gande R, Gibson KJ, Brown AK, Krumbach K, Dover LG, Sahm H, Shioyama S, Oikawa T, Besra GS, Eggeling L: Acyl-CoA carboxylases (accD2 and accD3), together with a unique polyketide synthase (Cg-pks), are key to mycolic acid biosynthesis in Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis. J Biol Chem. 2004, 279: 44847-44857. 10.1074/jbc.M408648200.PubMedGoogle Scholar
- Chami M, Andreau K, Lemassu A, Petit JF, Houssin C, Puech V, Bayan N, Chaby R, Daffe M: Priming and activation of mouse macrophages by trehalose 6,6'-dicorynomycolate vesicles from Corynebacterium glutamicum. FEMS Immunol Med Microbiol. 2002, 32: 141-147.PubMedGoogle Scholar
- Hard GC: Electron microscopic examination of Corynebacterium ovis. J Bacteriol. 1969, 97: 1480-1485.PubMed CentralPubMedGoogle Scholar
- Takayama K, Wang C, Besra GS: Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin Microbiol Rev. 2005, 18: 81-101. 10.1128/CMR.18.1.81-101.2005.PubMed CentralPubMedGoogle Scholar
- Yellaboina S, Ranjan S, Chakhaiyar P, Hasnain SE, Ranjan A: Prediction of DtxR regulon: identification of binding sites and operons controlled by Diphtheria toxin repressor in Corynebacterium diphtheriae. BMC Microbiol. 2004, 4: 38-10.1186/1471-2180-4-38.PubMed CentralPubMedGoogle Scholar
- D'Afonseca V, Prosdocimi F, Dorella FA, Pacheco LG, Moraes PM, Pena I, Ortega JM, Teixeira S, Oliveira SC, Coser EM, et al: Survey of genome organization and gene content of Corynebacterium pseudotuberculosis. Microbiol Res. 2010, 165: 312-320.PubMedGoogle Scholar
- D'Afonseca V, Moraes PM, Dorella FA, Pacheco LG, Meyer R, Portela RW, Miyoshi A, Azevedo V: A description of genes of Corynebacterium pseudotuberculosis useful in diagnostics and vaccine applications. Genet Mol Res. 2008, 7: 252-260.PubMedGoogle Scholar
- Dorella FA, Pacheco LG, Seyffert N, Portela RW, Meyer R, Miyoshi A, Azevedo V: Antigens of Corynebacterium pseudotuberculosis and prospects for vaccine development. Expert Rev Vaccines. 2009, 8: 205-213. 10.1586/147605188.8.131.52.PubMedGoogle Scholar
- Gordon D, Abajian C, Green P: Consed: a graphical tool for sequence finishing. Genome Res. 1998, 8: 195-202.PubMedGoogle Scholar
- Tauch A, Kaiser O, Hain T, Goesmann A, Weisshaar B, Albersmeier A, Bekel T, Bischoff N, Brune I, Chakraborty T, et al: Complete genome sequence and analysis of the multiresistant nosocomial pathogen Corynebacterium jeikeium K411, a lipid-requiring bacterium of the human skin flora. J Bacteriol. 2005, 187: 4671-4682. 10.1128/JB.187.13.4671-4682.2005.PubMed CentralPubMedGoogle Scholar
- Schneider J, Vorhölter FJ, Trost E, Blom J, Musa YR, Neuweger H, Schatschneider S, Tauch A, Goesmann A: CARMEN: In silico reconstruction of organism-specific metabolic networks using SBML. Genet Mol Res. 2010, 9: 1660-1672. 10.4238/vol9-3gmr901.PubMedGoogle Scholar
- Gough J, Karplus K, Hughey R, Chothia C: Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure. J Mol Biol. 2001, 313: 903-919. 10.1006/jmbi.2001.5080.PubMedGoogle Scholar
- Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, et al: The Pfam protein families database. Nucleic Acids Res. 2010, 38: D211-222. 10.1093/nar/gkp985.PubMed CentralPubMedGoogle Scholar
- Eddy SR: Profile hidden Markov models. Bioinformatics. 1998, 14: 755-763. 10.1093/bioinformatics/14.9.755.PubMedGoogle Scholar
- Dodd IB, Egan JB: Improved detection of helix-turn-helix DNA-binding motifs in protein sequences. Nucleic Acids Res. 1990, 18: 5019-5026. 10.1093/nar/18.17.5019.PubMed CentralPubMedGoogle Scholar
- Tatusov RL, Galperin MY, Natale DA, Koonin EV: The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000, 28: 33-36. 10.1093/nar/28.1.33.PubMed CentralPubMedGoogle Scholar
- Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25: 3389-3402. 10.1093/nar/25.17.3389.PubMed CentralPubMedGoogle Scholar
- Beckstette M, Homann R, Giegerich R, Kurtz S: Fast index based algorithms and software for matching position specific scoring matrices. BMC Bioinformatics. 2006, 7: 389-10.1186/1471-2105-7-389.PubMed CentralPubMedGoogle Scholar
- Baumbach J, Wittkop T, Kleindt CK, Tauch A: Integrated analysis and reconstruction of microbial transcriptional gene regulatory networks using CoryneRegNet. Nat Protoc. 2009, 4: 992-1005. 10.1038/nprot.2009.81.PubMedGoogle Scholar
- Crooks GE, Hon G, Chandonia JM, Brenner SE: WebLogo: a sequence logo generator. Genome Res. 2004, 14: 1188-1190. 10.1101/gr.849004.PubMed CentralPubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
Dreadlocks Islands and Skies edition book launch
Event date and time:Friday, February 15, 2013 - 12:00 - 12:30
The launching of a special "climate change and creativity"edition of Dreadlocks will take be hosted at the new Pacific Media Centre office in the Sir Paul Reeves Building next week.
It is the last edition of this publication as a discipline-based journal and the first as its revival as a multidisciplinary publication.
Dreadlocks was originally published as the literary journal of the School of Language, Arts and Media at the University of the South Pacific (USP), by the Pacific Writing Forum (PWF).
This special issue of Dreadlocks includes the proceedings of the Oceans, Islands and Skies - Oceanic Conference on Creativity and Climate Change – The Role of Writers, Artists and the Media in Environmental Challenges in the Pacific.

subset: commoncrawl


text:
(Disclaimer: This article contains words and phrases that some people may find offensive. Please do not continue if easily offended)
|© successyeti.com
"That mother-fucker STILL hasn't got back to me!"
Good morning, day or evening, inter-peeps. How many times have you torn yourself into little pieces over such words? I'm not just talking about that script you sent over to someone, that actor who liked your idea, that peer who couldn't be more up for it or that producer who was all up in your junk over a project. I'm talking about the many energy vampires we seem to stumble across throughout our filmmaking journey. The "Talkers".
Film noir street choked with fog.
Planted under a shaft of light -- A SILHOUETTE. Man, Woman or Beast. Nobody knows. Just then, JOE FILM-AKER cuts across the damp, cobbled street.
Nods on approach, eyes unable to make out the murky figure.
Hey, Man.
Good to finally meet you.
Yessssss. You tooooo.
So... You up for it?
Great! Look forward to hearing from you!
Joe walks away, content.
SHADY steps out, where a slit of light strikes across a pair of...
A little dramatic. But hey...
I personally take people on face value. When I chat/meet with someone, I try to gauge that person based on three things:
- How well do we get on
- Do we share any common interests
- Could we at some point work together in some capacity
As long as these boxes are ticked in my head, I'll take most of what they say as gospel. Not because I'm naive. Simply because I like to treat people the way I'd like to be treated. With respect and in good faith.
Communication is Key
If you're unable to commit to something, make it known. Email, text, a fucking carrier Pigeon... Whatever floats your boat. The worst thing you can do is go radio silent (something I've been guilty of in the past). This to me says two things:
- You're unreliable
- You're not worth my time
Save the life and work excuses.
Everyone has important matters they need to prioritise on a day to day basis. Don't be an Amy, Tom, Dick or Ahmed (metaphorically speaking). Be invaluable. Be a Walker, not a Talker. (thought I wrote wanker there for a minute! Hahahaaa, dyslexic much).
We're all on a slippery creative road, no matter what level we're at. The easier we can make it for each other, the more traction we can potentially gain.
Until next time, folks.
You might also like:
Great vs Undeniable
Fight Your Demons
5 Things To Consider Before Quitting Your Day Job
e: bulentfilms[at]gmail.com


text:
Additional Information
|Bird Size
|XSmall, Small
|Bird Species
|Lovebirds, Budgies, Plum-Headed Parakeet , Cockatiels, Ringnecks, Lorikeets, Quakers, Conures, Hahns Macaw, Caiques, Rosellas
|35cm long (this is straight, so without the bend in wire)
|Naturally Wild
Great for chew machines! Review by Loren
Quality Price Value
I bought this product specifically for my female budgie. She chews everything and was taking to chewing her dowel perches in the cage, which frustrated me constantly buying perches!
She took to it straight away, I've had it a few months now and it's still going strong. My little termite chewer is very happy and keeps her occupied whilst I'm working. She even hangs from this and chews away. It is very entertaining to watch. I come home and wood shavings are on the cage floor so I know what she has been up to all day lol!
Great product! (Posted on 2/04/17)


text:
Quarq DFour 11R Road Carbon Power Meter Crankset 110mm GXP 175mm Black
The Quarq DFour isn't just changing the game for power meters its taking them to the next level. Based of the DZero model, but with some upgrades the DFour is made to fit shimano's latest generations of 11- speed road chainrings. The DFour has five strain gauges strategically placed throughout the spider in order to measure the Newton meters you're putting down to the pedals. It includes the AxCad accelerometer, which provides cadence information without any additional sensors or magnets. The DFour is also compatible with training software suites including Training Peaks WKO+, Training Peaks, Golden Cheetah, and Strava.
- Bike type: road
- Power Balance.
- Accuracy
Check out our FAQ page for answers to common order questions.
You want more information? Please contact us and ask!
Contact us
We strive to answer all emails within 24 hours.
7 a.m. - 7 p.m. MST
1199 West 2425 South
Woods Cross, Utah 84087
Chat / Email Support
Online Hours: Weekdays, 7 a.m. - 7 p.m. MST
In-stock items ordered by noon, Mountain Time, leave our warehouse the same day. Items ordered after that time will ship next business day. Look under the product price above to check the product's availability.
Within the continental United States
Standard ground: Orders of single or multiple products from Bikewagon.com ship to anywhere in the continental (lower 48) United States for $5.00. Orders over $50.00 will be shipped standard ground for free. Individual items labeled with free shipping will also ship for free.*
Express shipping: For an additional charge, orders may be shipped express and will typically arrive within 1 - 2 days. To apply this option, select "Express Mail" shipping at checkout.
Hawaii & Alaska
Shipments to Alaska and Hawaii are limited to those items that can be shipped via USPS mail.
International shipping
International shipments are limited to those items that can be sent via international mail. Some brands restrict sales to U.S. customers only. International customers are responsible for all customs fees.
While most international orders will arrive within two to three weeks via First-Class International mail, please be aware that some orders may take up to 8 weeks due to slow customs processing in some countries. To avoid the possibility of this, please select another shipping method at checkout.
For orders outside of the United States and Canada over $400, we require a wire transfer payment before shipping. If you are interested in making such a purchase, please contact us for details.
See our shipping policy for complete details and a list of restricted brands.
*Complete bikes, frames, wheelsets and other oversized items are excluded from $5 and free shipping and will include the shipping charge as indicated at checkout. High-quantity orders of multiple products may also include a shipping charge. International orders and orders going to Hawaii, Alaska and U.S. territories are also excluded.
**Additional charges will be applied to items shipped outside the continental U.S. International customers are responsible for all customs fees.
***Brands excluded from international shipping: Boblbee, BodyGlide, Brooks, CamelBak, Chariot, Continental, Five Ten, GoPro, Kuat, Look, MET, Native Eyewear, Newgy, Ortlieb, Polar, Rab, RACE, Racktime, SE Bikes, Sea to Summit, Sidi, Smith Optics, Teeter HangUps, Time, Tubus, Vittoria
If, for any reason, the product you purchase doesn't work out, we're happy to take it back!
Just return the item within 90 days of the original purchase and we'll refund you're money.
Be aware that -- depending on the reason for the return -- shipping costs may or may not be included in the final refund.
See our complete return policy for all the juicy details.
If you need to exchange an item for a different color or size, we're happy to facilitate your exchange. However, please note that all shipping costs incurred in the exchange are the customer's responsibility.


text:
quinta-feira, 2 de junho de 2016
7 TIP´s for P&L quality - Controllership
A good management information system should disclose a results statement - P & L containing among others the following topics:
1 Detailed information on the company's income statements as a whole and by business unit or segment, so if the company is structured. The concept of business unit involves treating certain segments as if they were separate businesses, where revenues are shown, costs, expenses, profit or loss, assets and liabilities.
2- The information must be demonstrated in comparison with previous year and budget (budget). Some companies beyond the Budget include a line for the Forecast (Forecast), which is a kind of Budget revised. The changes must be shown as a percentage and / or value and must be justified if they are significant. Usually put on their income statements or P & L column containing commentary and analysis on each line of the statements.
3- The Income Statement (P & L) should be well detailed enabling the identification of variable and fixed costs and expenses. A line for contribution margin should be included. This line will be represented after deduction of all variable expenses and costs. Must be shown columns with absolute values and percentages.
4 Costs must be demonstrated, separated in fixed and variable. Categories groups should facilitate the identification of raw materials, packaging, direct manufacturing costs, indirect manufacturing costs and other relevant costs. Expenses Depreciation and amortization should be shown separately.
5. Is important to incorporate several non-financial indicators such as volumes, production, inventory turnover, accounts receivable, etc. I've seen accounting systems where some non-financial information was reported managerially which constituted an important management information.
6 Unitary sales price and costs can be calculated by taking the total value divided by volume.
We reported total revenue and the total volume of tons sold, for example. We can divide total revenue by the volume calculating unit sales price per ton. It is important that the total revenues and volumes can be demonstrated by product and by customer.
7. Demonstrate expenditure groups, such as administrative and commercial. Show consolidated data that can be opened and detailed by types of expenses. Usually the income statements expenses are shown in large groups, such as administrative expenses, human resources, etc. It is important that the costs demonstrated by groups can be opened in different ways, as for account, for cost center, etc.
Free partial, simple and diretct translation from: http://blogdoprofessorari.blogspot.com.br/2014/04/fechamento-de-resultado-mensal-com.html
Postado por Ari Lopes às 08:02:00
Marcadores: Accountancy, Controllership
Assinar: Postar comentários (Atom)


text:
Hi there! Remember me?
I've been asleep since my last post! Well, either that or at work, or traveling on my never-ending trip! I committed to the traveling pre-pregnancy, and boy did it wear me out!
My house is still in recovery. But I am starting to wake up. Here are some pictures from my trips!
Indianapolis- Kate, Jennie and I had so much fun presenting our poster! It was very well received.
I ate this whole steak.
We went to this fun pizza place the night I got to Boulder! I was pretty sick from the plane, but I ate a good deal of mine, and it was pretty tasty!
And yes, there was snow on the mountains. The weather in Boulder was actually pretty warm, but since I left they've been covered in snow again!
I haven't done any knitting since I finished the granny square blanket. I've just been too tired. And my house is messy and completely overwhelming. I am hoping that once the semester is over, I will be able to coheres Austin into helping me do so e deep cleaning, and then maybe I will feel more like knitting…
To be honest, the thought of knitting for the bug has been overwhelming me. This week marks the end of my first trimester and I am feeling more comfortable in my skin. I spent the entire past twelve weeks either terrified or attempting not to think about it. I was so scared. I mean, utter terror. It's over. I made it. And I saw my doctor today, we listened to that little bug heart beat, and everything is the way it's supposed to be! And the fact that I'm still awake at 10 pm without even a nap, and that I've folded laundry and messed around on the Internet is kind of amazing ( did I mention that I've been asleep for the past month?)
And by the way, I wrote this on 23 April and then promptly fell asleep without publishing! Yay for sleep!


text:
Tag Archives: recipes
February 14th is just around the corner, so why not do something special this year? Nothing says romance like putting your own energy and attention into a delicious, home cooked, Paleo Valentine's Day meal. We'll help you with the concept and recipes; you tell your Valentine to get ready for a wonderful surprise.
It should go without saying, but make sure you create an intimate ambiance, including nice linens, soft lighting, candles, and music. Sit your Valentine down, raise your glasses with an organic, sulfite-free wine, and explain that tonight's meal has three themes, each of which reflect upon your relationship.
- 1 and ½ pounds pork belly
- ¼ purple cabbage, sliced thinly
- 1 large carrot, cut into bite-size pieces
- 1 onion, diced
- 1-inch piece of ginger, peeled and sliced thin
- 3 cloves garlic, pressed
- 1 tablespoon cumin
- 1 tablespoon oregano
- 1 tablespoon paprika
- Freshly milled black pepper
1. Put the pork belly pieces in the slow cooker with about 1 cup of water. Turn on high setting for about 2 hours.
2. Remove the pork belly pieces from the slow cooker, reserving the cooking water.
3. Bring a sauté pan to medium heat and cook the pork belly pieces until they are browned on all sides.
5. Return the browned pork pieces to the slow cooker with all remaining ingredients.
6. Cook on high setting for about 2 additional hours or low setting for 4 hours.
- ¼ cup walnuts
- 1 large bundle of cilantro, washed and roughly chopped
- ¼ cup olive oil
- ½ clove garlic, pressed
- 1 lemon, juiced
- Freshly milled black pepper
- Your choice of salad greens and vegetables for salad
2. Start adding the cilantro, about 1 handful at a time, processing well.
3. Add the black pepper, taste, and adjust the flavors, adding more lemon if necessary.
- ¾ cup dates, pitted
- 1 cup coconut milk
- 2 eggs
- ¼ cup cacao powder
- ¾ cup frozen raspberries
- Desiccated coconut, for garnish
1. If the dates are very dry, soak them for about 20 minutes in the coconut milk.
2. Using a mini food processor, process the coconut milk and dates until you have a smooth mixture.
3. Mix in the eggs and cacao powder
4. Stir in the frozen raspberries, but don't process them (leave them whole).
5. Pour the mixture evenly into four ramekins.
6. Position three or four raspberry halves on the surface of each ramekin after filling with the mousse mixture.
7. Place the ramekins in a baking dish with about 2 inches of boiling water.
8. Bake at 350°F for 15 minutes or until the mixture has firmed up. Alternatively, you can cook in the slow cooker for about 45 minutes, again with 1 to 2 inches of boiling water on the surface of the slow cooker.
9. Garnish with desiccated coconut to serve.
Christopher James Clark, B.B.A.
Nutritional Grail
Christopher James Clark, B.B.A. is an award-winning writer, consultant, and chef with specialized knowledge in nutritional science and healing cuisine. He has a Business Administration degree from the University of Michigan and formerly worked as a revenue management analyst for a Fortune 100 company. For the past decade-plus, he has been designing menus, recipes, and food concepts for restaurants and spas, coaching private clients, teaching cooking workshops worldwide, and managing the kitchen for a renowned Greek yoga resort. Clark is the author of the critically acclaimed, award-winning book, Nutritional Grail.
As winter nears and the days grow cold, a hearty, warming dinner begins to sound more and more inviting. Aren't Paleo and hearty polar opposites? Substitutes for white potatoes and beans surely cannot compete with Paleo.
The key is to focus on the flavors and textures of the abundant seasonal produce and pair it with whichever wild proteins we can procure most easily. Then, we add our favorite herbs or spices, throw them into the slow cooker or Dutch Oven, and let time do its work.
By incorporating proteins, veggies and fats into one dish or pot, we save time and cleanup and end up with a meal for the whole family that is not only in keeping with True Paleo, but is also tasty and satiating, without filling you up so much you feel like you need to roll into bed.
How do wild game chili or pulled pastured pork sound? Slow cook one, and Dutch oven the other. Mix and match modalities, proteins, and veggies. Thankfully this fork in the road doesn't have a "wrong way!"
A lovely wild game chili meal, best made the day before serving to allow flavors a chance to develop.
- 1 tbsp coconut oil
- 1 lb free range venison boneless loin, cubed
- 1 lb free range ground bison
- 1 medium yellow onion, chopped
- 4 garlic cloves, smashed
- 1 jalapeno pepper, minced (omit seeds and pith for less heat)*
- 2 cups homemade chicken or beef broth
- 1 cup red wine*
- 1 large, ripe tomato, crushed*
- 1 tbsp ground cumin
- 1 tbsp paprika*
- 1 small bunch fresh cilantro and/or scallions, chopped finely, including stems
- Lime wedges, for garnish
- 4 cups wild mixed green lettuces of your choosing
2. Add cubed loin and sear on each side until browned, then remove from pot.
3. Add ground bison and cook, stirring, until browned, and then remove from pot.
4. Add onion to pan and sauté until soft, about 5 minutes.
5. Add garlic and jalapeno and cook one more minute.
6. Add broth and wine and bring to boil, using wooden spoon to scrape any browned bits stuck to bottom.
8. Cover and reduce heat to low and simmer one hour, stirring occasionally.
9. Add cumin and paprika and simmer one hour longer.
10. Serve hot with cilantro or scallions and lime wedges on top, with greens placed in a mound on top of each bowl.
A fattier cut from a naturally raised animal is perfectly paleo, not to mention delicious, from time-to-time. Make this at least a day ahead to experience the full decadence.
- 2 tbsp coconut oil
- 3 lbs pastured pork shoulder
- 1 large sweet onion, chopped
- ½ small pineapple, cored, peeled and cut into chunks
- 2 large, ripe red tomatoes, halved*
- 1 cup dry white wine*
- 1 – 2 cups homemade chicken broth
- 4 cups organic baby kale
2. Place pork in oven and brown for two minutes on each side.
3. Remove pork from oven and place on plate, covered.
4. Add onion to oven and sauté five minutes.
5. Add pineapple, tomatoes and pork.
6. Pour in wine and broth (use more if you prefer more liquid, less if more of a stew).
8. Remove from heat, let cool completely and refrigerate overnight.
9. The next day, place the oven over a medium flame and cook one more hour; pork will easily pull apart with two forks.
10. Shred all pork with two forks and serve on a bed of raw baby kale to enjoy the jus.
Lorrie Cordain's Roasted Wild Colorado Pheasant and Blue Grouse, The Cordain Kitchen
Pining for a new culinary addition to your dinner menu? Leave the seeds for the birds and mix up your poultry creations with this protein-packed Paleo Diet recipe.
Serves 3-5
- 1 wild pheasant
- 1 wild blue grouse
- 4 large oranges, juiced
- 4 garlic cloves, pressed
- ¼ fresh sweet onion, chopped
- 2 sprigs fresh thyme leaves, stems removed
1. In 4 cup measuring container, combine orange juice, garlic, onion, and thyme.
2. Pour over birds and coat thoroughly.
3. Refrigerate for 30-60 minutes.
4. Preheat oven to High Broil.
5. Place birds on foil covered broiler pan and broil on high 5-8 minutes.
6. Turn birds and broil for an additional 5-8 minutes.
8. Remove from oven and baste one final time before serving.
9. Serve with locally grown, sauteed organic broccoli, grilled mini peppers, onions and garlic in olive oil with dried, salt-free spices until tender.
10. Bon Appétit!
Live Well, Live Longer.
The Paleo Team
Each year, October 31st seems to mark the start of the junk food season. Most of us have fun childhood memories of dressing up in costume, attending parties, and spending hours Trick-or-Treating in our neighborhoods. Returning home with a bag full of candy and sugary snacks meant an evening spent sampling the hard, soft, or chewy treasures we had collected. Unfortunately, most of us probably went to bed with a tummy ache and awoke within a few days with a sore throat, cold, or flu virus due to the negative impact on our immune systems after overindulging. It occurred to me years ago that the cold and flu season really seems to get ramped up and into high gear beginning around Halloween and continuing well into Spring when the junk food holidays finally end at Easter time.Why not try out a Paleo Halloween this year and see where it can lead?
So, what's a Paleo parent to do? How do we help our children enjoy the festivities and traditions of our special holidays, while maintaining healthy eating habits? Over the past 10 years or so, there has been more and more awareness regarding these issues. Conscientious parents have tackled this problem in a variety of ways. Some have done away with Halloween altogether, while others have found new and creative ways to celebrate the day and start new, healthy traditions. Fortunately, the internet has become a fantastic place to search for special, healthy treats. Just last week, I found a darling creation making ghosts from ½ banana using small currants or raisins for eyes and mouth. Another site demonstrated making "Jack-o-lanterns" from oranges, carving out the skins to make the face. Check out just a few of the links to help you and your children get started. Be sure to include the kids in the search and make it fun and exciting to plan for your special day.
But, what to do with all that Trick-or-Treat candy? Again, it's time to get creative and think outside the box. Several approaches to this dilemma should be considered. Some parents allow their children to choose a small amount of candy to enjoy as a special treat over the next few days, throwing the rest in the trash where all the junk belongs. Others, strike a deal with their kids and agree to buy all of the candy from the child. The child is then allowed to spend the money on a toy or other non-food item of their choice. We know of a family who puts on a wonderful, healthy costume party for their children and their friends every year. There is no Trick-or-Treating, just healthy snacks, traditional games like bobbing for apples, and a fun time for all. If you don't want to throw your own party, many community organizations offer alternatives for kids. As for what to throw in those sacks when the neighborhood goblins come calling?
How about this treat: The Paleo Diet Bar in either Cinnamon Raisin, or Cranberry Almond! This is a special and fun time of year when lasting childhood memories are created. Start your own family traditions and enjoy the celebrations!
All the best,
Lorrie Cordain, M.A.
The Paleo Diet Team
Baked Apple
The aromas of this simple-to-prepare fall treat fills your house with sweetness and will have your kids drooling. Be sure to choose locally grown, organic apples when preparing this sweet treat.
- 1 large, fresh pie apple
- 1 medium orange, juiced
- 1 tbsp raisins
- ¼ tsp cinnamon
- Pinch allspice
- olive oil
1. Preheat oven to 350°F
2. Core the apple
3. Mix orange juice, raisins, cinnamon, and allspice together in small bowl and fill the cavity of the apple
4. Pour remaining liquid over apple
6. Bake 30-40 minutes or until soft
7. A small paring knife should easily puncture the apple skin to cavity center
8. Better get lots, the little goblins will certainly be begging for more!
Roasted Pumpkin Seeds
From jack-o-lanterns to pies, pumpkins have become an American tradition during this time of year. But, what to do with all those seeds after carving your spooky creation? This simple recipe has become a favorite of Paleo dieters everywhere.
4-6 Servings
- 1 large pumpkin
- 2 tbsp extra virgin olive oil
- 1 tbsp paprika
- 2 tbsp garlic powder
- 2 tbsp onion powder
- 1 tsp chili powder, to taste
1. Preheat oven to 350°F
2. Carve pumpkin, remove seeds
3. Thoroughly rinse pumpkin from seeds and pat dry with paper towels
4. Put seeds in a bowl and mix thoroughly with olive oil and spices
5. Spread seeds evenly on non-stick baking pan
6. Bake for 30 minutes, turning seeds every 10 minutes
Dr. Cordain's protein packed and flavorful scrambled eggs are guaranteed to jump start your energy levels for the day. Healthful fats and nutrients combined, this dish is one no Paleo breakfast menu should be without.
Serves 1-2
- 3 Cage-free, non-antibiotic/hormone eggs
- 1/4 – 1/2lb Breakfast sausage, ethically-raised, preservative-free
- 3 Bacon Strips, preservative-free
- 1/2 Onion, diced
- 1/2 – 1 cup Fresh green chilli or salsa (optional if you are avoiding nightshades)
2. Layer bacon, sausage bits, and diced onions in a small, non-stick 6-inch frying pan.
3. Stir frequently with a wood spatula for 8-12 minutes or until cooked evenly.
4. Whisk three eggs in a small mixing bowl.
6. Stir frequently to avoid burning for 2-4 minutes or until eggs are set.
7. Plate egg scramble and garnish with fresh green chili or salsa
Live Well, Live Longer.
The Paleo Team
The cookbook based on the bestselling The Paleo Diet.
Dr. Loren Cordain's The Paleo Diet has helped thousands of people lose weight, keep it off, and learn how to eat for good health by following the diet of our Paleolithic ancestors and eating the foods we were genetically designed to eat. Now this revolutionary cookbook gives you more than 150 satisfying recipes packed with great flavors, variety, and nutrition to help you enjoy the benefits of eating the Paleo way every day.
- Based on the breakthrough diet book that has sold more than 100,000 copies to date.
- Includes 150 simple, all-new recipes for delicious and Paleo-friendly breakfasts, brunches, lunches, dinners, snacks, and beverages.
- Contains 2 weeks of meal plans and shopping and pantry tips.
- Features 16 pages of Paleo color photographs
- Helps you lose weight and boost your health and energy by focusing on lean protein and non-starchy vegetables and fruits.
- From bestselling author Dr. Loren Cordain, the world's leading expert on Paleolithic eating styles.
Put The Paleo Diet into action with The Paleo Diet Cookbook and eat your way to weight loss, weight control maintenance, increased energy, and lifelong health-while enjoying delicious meals you and your family will love.


text:
NECBrochure: The NEC brochure provides a preliminary introduction to deafblindness, the New England Center, and eligibility criteria. (PDF
Infant Brochures:
The Vision and Vision & Hearing Brochure
provide information on vision and hearing impairment, sensory development, specialty providers, and Early Intervention. (PDF)
National Center on Deaf-Blindness Document Resources: The National Center on Deafblindness maintains a library of resources that can be helpful to families and personnel working with individuals who are deafblind.
To find items on the NCDB server click here to search:
Charge Syndrome Fact Sheet - Information about children with CHARGE Syndrome
Parent Road Map- a guide to raising a child with combined hearing and vision loss.
Parent Road Maps for:
Students with Cochlear Implants:
These guidelines are designed to facilitate discussion among professionals and families for the purpose
of monitoring educational programming and supports for communication access for students with cochlear
implants. This document is a tool to assist in the development of a student's Individualized Family Service Plan (IFSP),
Individualized Education Program (IEP), 504 plan, or other educational planning document with the goal
of complementing comprehensive assessments of language, communication, and other
competencies that influence programming decisions.
Guidelines for Educational Program Planning
Innovation Configuration for Evidence-Based Practices for Students With Sensory Impairments
The test of any intervention or procedure is evidence—not "whatever works" but "what works." There may be more information about the effectiveness of various consumer products than the methods we use to teach children and youth with sensory impairments.This paper features an innovation configuration (IC) matrix that can guide teacher preparation professionals in the development of appropriate content for evidence-based practices (EBPs) for students with sensory impairments.
For more information, please click here
Directions for the New Hampshire Communication Plan for a Student who is Deaf or Hard of Hearing
A communication plan needs to be completed for any student with a hearing loss who is being considered for or is receiving special education and related services. Students for whom hearing loss is a secondary disability, or who have concomitant visual impairments are included. The Communication Plan may also be helpful in planning for services for students with hearing loss under Section 504.
CADRE Conference 2015 Session Materials
Just Launched: Journal of Deafblind Studies on Communication
This journal foregrounds knowledge that is developing in a new academic study-line that focuses on communication and deafblindness from various theoretical perspectives. The study-line is connected to the International Master in Pedagogical Sciences on Communication and Deafblindness at the University of Groningen in the Netherlands.


text:
When you are tested for HIV, there are multiple conditions that can make the test result positive when in fact you DO NOT have AIDS.
The following list are just SOME of the factors known and proven by the medical community, to cross-react with the test and cause false/ inaccurate Positive HIV Antibody test results, for the complete list please refer to SOURCE. This information is from the most authoritative study yet written on AIDS. Available from Amazon and the author`s website
AIDS,OPIUM, DIAMONDS,and EMPIRE by Nancy Turner Banks,MD.
Factors known to cause false-Positive HIV Antibody test results :
– anti-carbohydrate antibodies
-naturally-occurring antibodies
-systemic lupus erythematosus
-renal (kidney) failure
-flu vaccination
-herpes simplex I
-herpes simplex II
–upper respiratory tract infection
-recent viral infection or exposure to viral vaccines
-pregnancy in multiparous women
-rheumatoid arthritis
-hepatitis B vaccination
-tetanus vaccination
-acute viral infections, DNA viral infections
– malignant neoplasms (cancers)
-alcoholic hepatitis/alcoholic liver disease
-visceral leishmaniasis
end of quote.
Many of the above conditions are common in Africa.


text:
Habits change in the fall these changes and these pattern movements in fall fishing occurs more in how the largemouth bass changes their habits, and why they relate to open flats more than any time of the year. Reason is simple, bass will more often follow the forage or bait fish than any other time of year; this can make them harder to locate as most anglers are use to looking for structure to hold them.
The lakes are always full of bait fish in the fall and bass move to the baitfish for easy feeding and ignore their tendency to hang on structure. Point being is dont ignore open water points or movement even though it appears to be removed from grass lines or structure that you are traditional use to fishing. This is one of the biggest reasons that crank baits running from 5 to 8 feet of water are so effective in the fall. You can be fishing hundreds of yards off structure and catching fish in the fall just because the bait have moved to the open waters. Long extended points where youre removed from the traditional structure become great places to catch bass this time of year. Baits like rattle baits become once again effective, as open flats may not be covered in grass, and their tendency to chase becomes a pattern.
Have you ever wondered why in the fall it seems that the bass turn on and off like a light switch, the reason is very simple the bass feed in large groups in the fall? They feed two maybe three times a day, the rest of the day they suspend and become very lethargic in the warm water. Your fishing habits in the fall can many times be timed like clockwork as bass can be feeding one minute, your catching fish and then gone and not feeding the next. This is why I fish so many reaction type baits in the fall, because outside the feeding times the bass can be very hard to catch and the only way is to trigger their instincts to react.
Dont let the slow times of fall fishing discourage you, just look at some nontraditional areas away from structure, work reaction baits and you will find the bass.
Fish Lake Guntersville Guide Service
Email: firstname.lastname@example.com
Call: 256 759 2270
Captain Mike Gerry


text:
UDP-based Data Transfer Protocol (UDT)
is a high-performance data transfer protocol designed for transferring large volumetric datasets over high-speed wide area networks.
Key feaures include
Nimble Streamer has full support for UDT protocol.
To start using UDT in Nimble Streamer, you need to do the following:
Nimble Streamer covers wide variety of live streaming scenarios so UDT can be used as an element in complex delivery scenarios.
Nimble Streamer can be installed in a couple of small steps and upgraded to the latest version by running two or three simple commands in console.
Let us know if you need any help and search through our documentation to find more information.
This website or its third-party tools use cookies, which are necessary to its functioning
and required to achieve the purposes illustrated in the
Privacy Policy.
If you want to know more or withdraw your consent to all or some of the cookies, please
refer to the
Privacy Policy.
By closing this banner, scrolling this page, clicking a link or continuing to browse otherwise, you agree to the use of cookies.