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Genes 2011, 2(3), 640-660; https://doi.org/10.3390/genes2030640
Abstract: Pseudomonas syringae is pathogenic in a wide variety of plants, causing diseases with economic impacts. Pseudomonas syringae pathovars produce several toxins that can function as virulence factors and contribute to disease symptoms. These virulence factors include antimetabolite toxins, such as tabtoxin, phaseolotoxin and mangotoxin, which target enzymes in the pathways of amino acid metabolism. The antimetabolite toxins are generally located in gene clusters present in the flexible genomes of specific strains. These gene clusters are typically present in blocks of genes that appear to be integrated into specific sites in the P. syringae core genome. A general overview of the genetic organization and biosynthetic and regulatory functions of these genetic traits of the antimetabolite toxins will be given in the present work.
1. Introduction
Pseudomonas syringae is an opportunistic phytopathogenic bacterium that normally exists as an epiphyte, but if the host's defences are compromised, P. syringae becomes pathogenic. This species of bacteria infects a large variety of plants and produces a wide spectrum of phytotoxic compounds [1], and it is well adapted to survive in plant environments. The phytotoxins produced by P. syringae pathovars are not host-specific [2]. Although not essential for pathogenicity, they generally act as virulence factors and are involved in the production of disease symptoms in many plants [1,3]. Some of the most important phytotoxins produced by P. syringae are toxins belonging to the syringomycin group, which is a virulence determinant of phytopathogenicity. Syringomycin toxins consist of a polar peptide head with a lipophilic, fatty-acid tail; they have amphiphilic properties, which can lower surface tension and interact with and alter the integrity of cellular membranes [4,5]. The genes involved in the production of these toxins, including the non-ribosomal peptide synthetase (NRPS) systems, are mainly clustered in the bacterial chromosome [1,6]. For example, the syringomycin (syr) gene cluster of P. syringae pv. syringae strain B301D is about 37 kb in size, and it consists of two non-ribosomal peptide synthetases (NRPS; syrB1 and syrE); the syringopeptin gene cluster (syp), which is directly adjacent to the syr gene cluster, is 74 kb and carries 22 NRPS modules [7]. Another phytotoxin produced by several pathovars of P. syringae is coronatine. This virulence factor promotes entry of the bacteria into the plant host by stimulating the opening of the stomata [8] and suppressing the salicylic acid-dependent host defences [9,10]. Several adjacent gene clusters are necessary for the biosynthesis of coronatine, including the cor cluster (corPSR), which helps control the transcription of the cma and cfa clusters [11]. According to results obtained from the study of genes belonging to this cluster and are responsible for the biosynthesis of coronamic acid, which is non-proteinogenic, the involvement of a thiotemplate mechanism that could be located in the cma cluster is likely [12,13]. The polyketide portion of coronatine, coronafacic acid, is synthesized by the cfa cluster, which consists of nine open reading frames [14,15].
The antimetabolite phytotoxins, which are the subject of this overview, consist of small peptide molecules that interfere with the nitrogen metabolism of host cells and influence the course of disease development or symptoms. Although the antimetabolite toxins are not required for P. syringae to be pathogenic, they generally function as virulence factors, and their production results in increased disease severity [3,16,17]. The targets of all the described antimetabolite toxins are enzymes involved in the biosynthetic pathways of amino acids, such as glutamine or arginine. One of the better known antimetabolite toxins is tabtoxin, which is a β-lactam that inhibits glutamine synthetase (GS) (Figure 1), causing a glutamine deficit and an ammonium increment. Another well-known antimetabolite toxin is phaseolotoxin, which is a sulfodiaminophosphinyl peptide that disrupts the urea cycle by inhibiting ornithine carbamoyltransferase (OCT), causing arginine deficiencies. Mangotoxin, which is a more recently described toxin, inhibits ornithine acetyltransferase (OAT) (Figure 1), producing an ornithine deficit and interfering with arginine metabolism. Two other antimetabolite toxins, both of which inhibit the enzymes that catalyse the transformation of N-acetyl-glutamate into N-acetyl-ornithine, have also been detected. However, unstable precursors have hindered our ability to gain knowledge about the target enzymes of these last two antimetabolite toxins [18,19]. Studies on the phylogeny of antimetabolite toxins have concluded that toxin production is not irremediably linked to a specific pathovar or pathovars of P. syringae. Toxigenic and non-toxigenic P. syringae strains may belong to the same pathovar. Moreover, it is unusual to detect strains that produce more than one kind of antimetabolite toxin, so toxin production can be consider poorly associated with the host of isolation [20].
The genes encoding the antimetabolite toxins have not been found homologues outside of the P. syringae genome [20]. Study of the metabolism of toxins has led to the discovery of novel biosynthetic mechanisms, which typically involve non-ribosomal synthesis catalysed by multifunctional proteins or polypeptide complexes and intermediates that are transferred between enzymatic domains and not released into the cytoplasm. These enzymes are normally encoded in gene clusters, which are typically present in block of genes that appear to integrate into specific sites in the P. syringae core genome [7]. These gene clusters present qualities that are inconsistent with clonal (vertical) evolution. Contrarily, the evolution of toxin-producing genes is very likely driven by horizontal gene transfer [21].
The main goal of the current review is to present the research studies that have resulted in a better understanding of the genetic organization, biosynthetic pathways and regulatory functions of the three best-known antimetabolite toxins produced by pathovars of P. syringae.
2. Tabtoxin
Tabtoxin is a phytotoxic dipeptide produced by P. syringae pvs. tabaci, coronafaciens and garcae [22]. It is composed of tabtoxinine-β-lactam [2-amino-4-(3-hydroxy-2-oxoazacyclobutan-3-yl) butanoic acid] and either serine or threonine (Figure 2) [23]. It has been suggested that in planta, some peptidases convert tabtoxin to tabtoxinine-β-lactam (TβL), which irreversibly inhibits the target enzyme glutamine synthetase (GS). This inhibition results in the abnormal accumulation of ammonium, causing the characteristic chlorosis [24].
2.1. Resistance
The first detected gene related to tabtoxin was named the tabtoxin resistant gene (ttr); it is specific for the inactivation of tabtoxin and not for other inhibitors of glutamine synthetase, such as bialaphos and methionine sulfoximine [25]. To examine the function of the ttr gene, tabtoxin was incubated with [14C]-acetyl-CoA in crude bacterial extracts. Radioactive acetylated products were detected only in extracts of Escherichia coli transformed with a tabtoxin resistance gene, indicating that the ttr gene encodes an enzyme which acetylates tabtoxin or TβL [26]. In some works, it has been speculated that the acetylation probably occurs in a TβL amino group according to a mechanism similar to the inactivation of bialaphos [27,28]. To confirm this function of the ttr gene, Anzai et al. [26] transformed tobacco plants with the ttr gene, confirmed the integration of ttr into the genomic DNA of transgenic tobacco plants and tested its expression levels. When the transgenic tobacco plants were inoculated with P. syringae pv. tabaci, none of them produced the chlorotic halo typical of the wildfire disease seen in non-transgenic controls. This strongly suggested that the ttr gene expressed in tobacco plants provides resistance to the effects of tabtoxin [26]. Sometime later, the function of the ttr gene was confirmed, and the mode of action was described by analysis of the structure of the ttr gene product (TTR) to confirm that TTR is an enzyme that functions as an acetyl-transferase [29]. Tabtoxin-resistance protein TTR was determined to be an enzyme that renders tabtoxin-producing pathogens tolerant to their own phytotoxin.
Meanwhile, Kinscherf et al. [30] continued searching for genes related to tabtoxin biosynthesis. By constructing Tn5-defective mutants, these authors characterized a DNA region highly conserved among tabtoxin-producing pathovars of P. syringae, which could be deleted at a relatively high frequency (10−3/cfu), in the BR2 strain of P. syringae. The cosmid pRBL823, which was obtained from analysis of Tn5 mutants, contains clones of about 32 kb of genomic DNA, and it can restore the production of tabtoxin in Tox− spontaneous deletion mutants; the cosmid even enabled tabtoxin production and resistance in the P. syringae epiphyte Cit7, which does not naturally produce tabtoxin, indicating that pRTBL823 contains a complete set of genes for the biosynthesis of and resistance to tabtoxin. However, when sequences containing the ttr gene were compared with the cosmid pRTBL823 by Southern blot analysis, no cross-hybridisation was found. According to their data, the ttr gene is located outside the cloned region, which does not support the conclusion that this gene is the only source of resistance to TβL exhibited by tabtoxin-producing strains. Moreover, their results indicate that tabtoxin is required by the P. syringae BR2 strain for both chlorosis and lesion formation in beans. However, toxin production was not required for growth in planta; until then, it had been impossible to genetically separate toxin production from the disease symptoms caused by this strain of P. syringae pv. tabaci [30].
2.2. Identification of the Genes Involved in Tabtoxin Biosynthesis
The study of approximately 3 kb of the cloned region revealed the presence of the genes tblA and tabA (also called tblB), which are required for tabtoxin production (Figure 3). The tblA gene encodes a product with an amino acid sequence unrelated to those of known polypeptides. This gene seems to be regulated by the lemA (now named gacS) gene via mRNA. The gene lemA (gacS) belongs to a family of two-component regulatory genes (gacA/gacS) involved in pathogenesis [31]. The regulation of the tblA gene may occur at the level of transcription; however, posttranscriptional effects, such as message stability, have not been ruled out.
Barta et al. [31] had no evidences that the tblA gene was part of a multigenic operon; on the contrary, mutations in genes upstream of tblA did not inhibit production of its transcript, nor did they prevent tabtoxin production. Furthermore, operon fusion data suggested the presence of a fairly strong promoter separate from the tblA gene [33]. The tabA product presented an amino acid sequence homologous to meso-DAP decarboxylase (the lysA gene product). The similarity between tabA and lysA suggests that the biosynthetic pathways of lysine and TβL could be related, however, tabA was unable to complement an Escherichia coli lysA mutant [32] The tabA gene is located immediately downstream of the tblA locus; however, the 1 kb transcript was not large enough to encode the predicted gene products of both the tblA and tabA genes. This could indicate that these two genes are transcribed separately. Their studies also showed that bacteria in minimal medium produced the tblA message during all growth phases. The tblA transcript was also detected in rich medium; however, the presence of other amino acids interferes with the detection of TβL by both bioassay and amino acid analysis [33], which is a common problem in the study of antimetabolite toxins.
2.3. Biosynthesis Proposals
Liu and Shaw [34] attempted to clarify the biosynthetic pathway of TβL by obtaining mutants of the dapB gene from tabtoxin-producing strains. The dapB gene encodes l-2,3-dihydrodipicolinate (DHDPA) reductase, the second enzyme of the lysine branch of the aspartic amino acid family, and mutation of dapB produces diaminopimelate (DAP) auxotrophs and tabtoxin-negative mutants. DAP mutants were obtained from a parent strain (tabtoxin-producing strain) and the mutants in which the TβL biosynthetic locus was deleted. Restriction digestion and hybridisation analysis indicated that Tn5 insertion had occurred in the same locus in both the mutants and the parents. These data led to the conclusion that in in vitro conditions, the wild-type, tabtoxin-producing strain contained a single functional copy of dapB, a gene that was not located in the TβL biosynthetic region. In addition, this region did not contain a gene that encodes a protein with DHDPA reductase activity. In addition to requiring DAP and lysine for growth, dapB mutants also failed to produce TβL, indicating that neither DAP nor lysine is a tabtoxin precursor [34]. Moreover, results obtained from dapB gene complementation, coupled with the observed phenotype in dapB mutants, lead to the prediction that DapB was an enzyme required for the biosynthesis of both DAP and TβL and that tetrahydropicolinate (THDPA) was a common intermediate in those biosynthetic pathways [34].
A few years later, Kinscherf and Willis [35] published a study that revealed the biosynthetic gene cluster for TβL in Pseudomonas syringae. They performed an analysis of the tabtoxin gene transfer block, where they observed that the portion of the tabtoxin gene island closest to the lysC tRNA gene contain genes associated with genetic dynamism; even the gene adjoining the tRNA gene encodes a full-length product with high similarity to XerC (tyrosine recombinase). Such genes have often been found near the beginning of chromosome regions thought to result from the horizontal transfer of DNA into the current host [36]. The biosynthetic gene cluster contains the "leftward" block, which is separated from the "rightward" block by a 1 kb, AT-rich section that could be a bidirectional promoter (Figure 3, Table 1). The first gene product in the left biosynthetic block, as transcribed right to left, is TabC, which contains a motif homologous to some zinc-binding proteins but was otherwise novel in the database. The next gene product is TblA, which has weak similarity to methylase but was also novel, then TabA, which has high similarity to diamopimelate decarboxylase (LysA). TabB was highly similar to succinyldiaminopimelate aminotransferase (DapD), and TabD was an Aat-like aminotransferase. Finally, TabP is a putative zinc metallopeptidase that seems to be responsible for the maturation event that converts tabtoxin to its toxic form, tabtoxinine-β-lactam.
The first two genes in the right biosynthetic block (Figure 3, Table 1) encode proteins similar to a pair known to be involved in the synthesis of clavulanic acid. The first open reading frame (ORF) described, TblS, is similar to β-lactam synthase and is related to the asparagine synthetases that catalyse the formation of the β-lactam ring in clavulanic acid [37]. The adjacent ORF, TblC, resembles clavaminic acid synthase. TblD appears to represent an interesting gene fusion whose product is a full-length glucose-methanol-choline-oxidoreductase (GMC-oxidoreductase) with a GNAT family acetyltransferase domain on the carboxy-terminus. TblE is a putative membrane protein, and TblF, is presumed to be an enzyme with similarity to d-alanine-d-alanine ligase. The last ORF, which encodes a member of the transporter and multi-drug resistance, was designated as TblR [35]. Thus, tabtoxin shares part of the biosynthetic pathway of lysine (dabABCDE), branching off after tetrahydrodipicolinate but before diaminopimelate formation. From this branch point, tabtoxin is assembled by the actions of TblS, TblC, TblD and TblE in conjunction with TblF. Upon assembly, the completed tabtoxin can be converted by the metallopeptidase TabP into the toxin TβL, which is subsequently exported by the transporter TblR [7].
Knowledge of the gene cluster responsible for the biosynthesis of tabtoxin has allowed the development of other techniques and studies. For example, specific detection methods for tabtoxin-producing pathovars have been developed, as have PCR protocols that utilize oligonucleotide primers derived from the coding sequences of the tblA and tabA genes for the quick and reliable detection of tabtoxin-producing bacteria. This PCR detection method uses the primer sets to amplify a single 829-bp amplification product of tblA and another single 1020-bp amplification product of tabA, genes that proved to be specific for tabtoxin-producing organisms [38].
|Putative function
|NCBI gene id number
|Putative zinc binding protein
|AF519896.1 a
|Tabtoxin biosynthesis enzyme
|Meso-DAP decarboxylase
|Succinyldiaminopimelate aminotransferase
|Aat-like aminotransferase
|Zinc metallopeptidase
|β-lactam synthase
|Similar to clavaminic acid synthase
|Putative membrane protein
|D-Ala-D-Ala ligase
|nd b
|Multidrug resistance transporter protein
atabC, tblA and tabA genes are annotated together with the same accession number;btblF has not found in NCBI database.
3. Phaseolotoxin
Phaseolotoxin is a phytotoxic tripeptide consisting of ornithine, alanine and homoarginine that is linked to an inorganic sulfodiaminophosphinyl moiety (Figure 4) [16,39]. Phaseolotoxin is produced at relatively low temperatures (18 to 22 °C) by Pseudomonas syringae pvs. phaseolicola and actinidiae, pathogens causing halo blight in beans and canker in kiwi fruit, respectively. It is also produced by P. syringae pv. syringae CFBP3388 [40]. Phaseolotoxin can be cleft by peptidases and can liberate sulfodiaminophosphinyl ornithine (Psorn, also called octicidine [39]), the irreversible inhibitor of ornithine carbamoyltransferase (OCT), an enzyme that catalyses the conversion of ornithine to citruline.
3.1. Resistance
Initial studies on genes involved in the biosynthesis of phaseolotoxin using random mutagenesis by Tn5 insertion. This resulted in the localization of a KpnI genomic fragment of about 28 kb (including the Tn5 insertion), which restores the production of phaseolotoxin in defective Tn5 mutants. The pattern of restoration observed in the mutants and their plasmids evoked a cluster of genes initially called tox [41]. The first gene identified in the phaseolotoxin gene cluster was argK (Figure 5), which encodes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT), which prevents PSorn inhibition [42]. Hatziloukas and Panopoulos [42] conducted a study in which they established that the enzyme ROCT did not evolve from the housekeeping sensitive OCT (SOCT). This was consistent with the idea that argK is not derived from the argF gene, which encodes SOCT, of P. syringae pv. Phaseolicola. The argK gene must have immigrated into the organism's genome from another source by horizontal transference. The physical proximity of argK to the cluster of the genes involved in the biosynthesis of phaseolotoxin and its role in phaseolotoxin-resistance suggest that the entire argK-tox cluster was transferred en masse into the P. syringae pv. phaseolicola genome [42].
The argK transcription and regulation studies performed by Hatziloukas and Panopoulos revealed three argK-specific transcripts and a transcription start point near the putative ROCT initiation codon. The results of RNA sizing indicated that a small (presumably monocistronic) argK mRNA and two larger (potentially bi- or multicistronic) argK mRNAs exist in the cells (Figure 5). The argK promoter was stimulated in arginine-supplemented cultures at both high and low temperatures, but to a different degree. The greater activation of the argK promoter at 28 °C effectively cancelled the apparent repression of the argK promoter by elevated temperature in the minimal medium [42].
3.2. Identification of Genes Involved in Phaseolotoxin Biosynthesis
One year later, in 1993, Zhang et al. [43] published a study on the genetic organization of a cluster of genes involved in the production of phaseolotoxin. They showed that P. syringae pv. phaseolicola genomic clone pHK120, which complemented all their Tox− mutants, can be divided into at least eight complementation groups, phtA to phtH (Figure 5). Although pHK120 contains most of the genes involved in the production of phaseolotoxin, they concluded that it does not contain all of them, since pHK120 does not contain the ROCT gene (argK). The fact that transconjugants containing pHK120 produced toxin at 28 °C indicated that the thermoregulation of toxin production was overridden by pHK120. The plasmid may have contained an activator; overproduction of this activator could abolish thermoregulation. Another possible explanation is that some or all of the phaseolotoxin genes present in pHK120 harbour DNA binding sites which titrate the putative repressor protein produced by P. syringae pv. phaseolicola at 28 °C. This clone does not harbour a structural or regulatory gene involved in toxin production, but it contains DNA-binding motifs similar to those involved in binding proteins in other systems. On the basis of these results, the authors proposed that when present in multiple copies, the fragment harbouring DNA-binding sites titrates a repressor protein produced at 28 °C, thereby allowing toxin biosynthesis to occur [43,44]. Similar DNA-binding motifs were found in the upstream region of the argK gene, which is co-ordinately regulated with phaseolotoxin genes by temperature [42].
Based on what we now know, the production of phaseolotoxin occurs at low growth temperatures (18–22 °C) and is genetically controlled by a group of genes called tox according Peet et al. [41] or pht according to Zhang et al. [43] clustered in a region approximately 28 kb in size. The phaseolotoxin biosynthetic cluster is now called the argK-tox cluster because it includes argK [45].
The fatty acids desaturase gene (desI) (Figure 5, Table 2) was unambiguously identified and located at the 3′-end of the complementation group phtE in the middle of the argK-tox cluster. Genes placed downstream from desI were revealed, as desI spans a region essential for phaseolotoxin production. The link between the desI product and the biosynthesis of phaseolotoxin is not immediately obvious; however, if it is possible to establish a relationship between fatty acids desaturase, phaseolotoxin secretion at low temperatures and cell membrane fluidity [45], then desaturase action produces an increase in membrane fluidity to facilitate the phaseolotoxin secretion. The next gene identified in the argK-tox cluster was called amtA (Figure 5, Table 2), which encodes amidinotransferase. In fact, biochemical activity corresponding to an amidinotransferase was detected in P. syringae pv. phaseolicola extracts in previous studies [46,47]. The amidinotransferase activity detected was shown to catalyse the transfer of an amidino group from arginine to lysine to produce one molecule of homoarginine and one molecule of ornithine, both of which are precursors in the biosynthesis of phaseolotoxin. Mutants in amtA, defective in amidinotransferase activity, lost the ability to synthesize homoarginine and phaseolotoxin when grown in minimal medium at 18 °C [48]. According the results obtained by these authors, amtA was thermoregulated following a pattern similar to that obtained for argK.
|Putative function
|Ortholog genes a
|NCBI annotation
|Phaseolotoxin insensitive ornithine carbamoyltransferase
|Hypothetical protein
|Hypothetical protein
|Hypothetical protein
|Hypothetical protein
|Hypothetical protein
|phtF (argD)
|Ornithine aminotransferase
|Hypothetical protein
|Fatty acid desaturase
|Fatty acid desaturase
|Deoxycytidine triphosphate deaminase
|phtK (dcd)
|Deoxycytidine triphosphate deaminase
|txi19- txi18
|txi19- txi18
|Pyruvate phosphate dikinase PEP
|Hypothetical protein
|phtN and phtO
|Hypothetical protein
|HAD superfamily hydrolase
|L-arginine:lysine amidinotransferase
|Hypothetical protein
|phtS (cysC1)
|Adenylylsulfate kinase
|Hypothetical protein
|L-amino acid ligase
|ISPsy5 Transposase truncated
aGenes annotated in NBCI data base with 100% of coverage and identity with corresponding gene in Ps pv. phaseolicola NPS3121. Accession numbers of the P. syringae strains used in the preparation of this table are as follow: Ps phaseolicola 1448A (NC_005773), Ps phaseolicola MAFF302282 (AB237164.1) and Ps actinidiae Kw11 (AB217563.1).
Several years later, Aguilera et al. [49] carried out a functional characterization of the gene cluster involved in phaseolotoxin biosynthesis (the pht cluster). It is composed of 23 genes and is flanked by insertion sequences and trasposases, which agrees with the hypothesis of the horizontal transference of the phaseolotoxin cluster. They demonstrated that the mutation of 14 genes within the cluster results in a Tox− phenotype for 11 of them, from phtA to phtT (Figure 5, Table 2), while three mutants exhibited only low levels of phaseolotoxin production. The Tox− phenotype clearly indicates that all genes within this region encode proteins that are required at one or more of the different stages of phaseolotoxin production, such as synthesis, transport or regulation. The 23 genes are organized in five transcriptional units, two monocistronic and three polycistronic, with one overlapping a larger operon. Thus, phtL and argK result in two independent monocistronic units, one polycistronic operon comprising 11 genes, from phtA to phtK, harbouring an internal promoter upstream of phtD, and another large operon comprising genes from phtM to phtV [49] (Figure 5). The authors also observed that null-phtL mutants cannot transcribe the phtM operon, which suggested a regulatory role for the phtL gene product, but this had no effect on any of the other transcriptional units. However, their findings indicate that both the phtD and phtM operons were transcribed from very similar promoters, suggesting a common mechanism of regulation. This in turn suggested the PhtL did not have an effect on the regulation of the argK or phtA promoters [49].
After publication of the whole genome sequence of P. syringae pv. phaseolicola strain 1448A, which was deposited in a database for public access (NC_005773 in the NCBI database), Arai and Kino [50] localized an l-amino acid ligase in the PSPPH_4299 gene in the phaseolotoxin gene cluster. The PSPPH_4299 protein synthesized various hetero-dipeptides, but no homo-dipeptides or tripeptides were detected. It was found that homoarginine was usable as a substrate, but ornithine was not. Further investigation revealed that ATP and Mg2+ were essential for peptide synthesis, and that ATP was hydrolysed to ADP and phosphate. Serine, threonine and glycine were also usable as N-terminal substrates, and resulted in the synthesis of hetero-dipeptides containing basic amino acids. d-amino acids were not usable as substrates [50]. They concluded that they had found a novel l-amino acid ligase, the PSPPH_4299 protein, which is encoded by a gene in the phaseolotoxin biosynthetic gene cluster from P. syringae pv. phaseolicola 1448A, suggesting that the l-amino acid ligase plays a physiologically important role in the synthesis of a peptide phytotoxin.
3.3. Application of Phaseolotoxin Genes in Disease Diagnosis
The presence of specific genes for phaseolotoxin production gave rise to the development of detection methods like the PCR technique. Although this molecular method can detect strains that produce phaseolotoxin, there are non-toxigenic strains that are still pathogenic. In fact, studies of the argK-tox gene cluster in non-toxigenic strains of P. syringae pv. phaseolicola, which are commonly found in the bean fields of northern Spain, have revealed that these strains are undetectable by available methods of fast detection [51]. PCR analysis of three collections of strains showed the presence of three groups: toxigenic strains with the phtE and argK sequences, non-toxigenic strains with the phtE and argK sequences (argK-tox cluster) and non-toxigenic strains without the argK-tox cluster. González et al. [51] revealed that the presence of wild, non-toxigenic strains that cause halo blight but do not carry the argK-tox gene cluster represent a drawback to the methods used to detect P. syringae pv. phaseolicola, as these strains cannot be detected by these methods in dry seeds, and halo blight is spread by sowing contaminated seeds [51].
A similar study was performed by Rico et al. [52], who studied the incidence of phaseolotoxin-producing strains in beans with symptoms of disease. Almost 69% of strains of P. syringae pv. phaseolicola isolated in northern Spain were non-toxigenic. Indeed, the non-toxigenic mutants of P. syringae pv. phaseolicola were still pathogenic, and their virulence was comparable to that of the wild type, except that there was no formation of chlorotic halos. These results confirm that phaseolotoxin is a virulence factor. They also found that the majority of strains isolated contained avrPphF; Thus, they developed an avr-PCR method, which extends the range of detection of P. syringae pv. phaseolicola [52].
These and previous results have led to the conclusion that the argK-tox cluster was integrated into the genome of P. syringae by horizontal transference. Furthermore, it is possible to detect the argK-tox cluster in two pathovars of P. syringae (phaseolicola and actinidiae) that belong to different phylogenetic groups. Therefore, three hypotheses were proposed by Sawada et al. [53]: (1) The argK-tox gene cluster was introduced by horizontal gene transfer from the original organism into the genome of a common ancestor of pv. actinidiae and pv. phaseolicola. Afterward, as the ancestor evolved into pv. actinidiae and pv. phaseolicola, the argK gene evolved with the rest of the genome; (2) After pv. actinidiae and pv. phaseolicola separated from their common ancestor, argK was introduced by horizontal gene transfer from the original organism into either pv. actinidiae or pv. phaseolicola, from which the gene was introduced by a second horizontal gene transfer into the other; (3) After pv. actinidiae and pv. phaseolicola separated, argK was introduced from the original organism into pv. actinidiae and pv. phaseolicola by two independent horizontal gene transfers. They found that the argK gene was introduced very recently, in evolutionary terms, and so rejected hypotheses (1) and (2). Their phylogenetic analysis of the evolutionary mechanism of the argK-tox gene cluster showed that it had expanded its distribution over two pathovars by horizontal gene transfer, so the third hypothesis seems to be most likely [53,54]. To confirm the possibility of horizontal transference between an original organism and two different pathovars of P. syringae, they performed a comparative analysis of the argK-tox cluster and the regions bordering it in phaseolotoxin-producing strains. The authors presented studies which confirmed that the tox islands (containing the argK-tox cluster, the phtA-phtH genes and the argK genes) localized on respective chromosomes of pv. actinidiae and phaseolicola were exogenous DNA regions, and they also clarified that both pathovars were phylogenetically distant from each other. However, almost identical tox islands were confirmed to be present on their diversified chromosomes. They identified three coding sequence (CDs) encoding putative tyrosine recombinases at the left end of the tox islands. Given that some tyrosine recombinases have been reported to catalyse the integration/excision of genomic islands, the authors hypothesized that these tyrosine recombinases were involved in the horizontal transfer of tox islands [55]. These results suggested that the tox islands were organized in some unknown bacteria distantly-related to P. syringae and then horizontally transferred and integrated site-specifically and in the same direction into the respective homologous sites of the pv. actinidiae and pv. phaseolicola chromosomes. However, the fact that the relative positions of the tox islands on the respective chromosomes of pv. actinidiae and pv. phaseolicola were quite different implies that the tox islands integrated site-specifically into the respective chromosomes after the relative positions of the sites for their integration had changed due to genomic rearrangement [55].
3.4. Regulation of Phaseolotoxin Production
The special characteristic of phaseolotoxin is that it can be produced at relatively low temperatures, which has allowed important studies to be conducted on the thermoregulation of genes involved with phaseolotoxin production. In fact, evidence obtained by Rowley et al. suggests that a protein produced by P. syringae pv. phaseolicola at 28 °C that binds specifically to both the thermoregulatory region (TRR) and argK fragments could be involved in the negative regulation of the argK gene in vivo at this temperature [56]. First, motility shift assays showed that there was a protein, detected only in cells grown at 28 °C, that binds specifically to both the TRR and argK fragments. Second, cross-competition data showed that the TRR fragment efficiently and specifically competes with the argK fragment for binding of the protein. Third, the sites in the argK fragment that were protected from DNAse I cleavage harbour conserved or semi-conserved core motifs found in the TRR [56]. Rowley et al. [56] sequenced the largest locus (phtE) (Figure 5) in the phaseolotoxin gene cluster; however, this operon is not regulated by temperature. Afterward, the sequences of six additional loci in the toxin cluster were analysed, and the results showed at least two genes harbouring sequences that presented similarity to the core motif [56].
Sometime later, López-López et al. [57] proposed a working model for the induction of genes involved in phaseolotoxin synthesis in P. syringae pv. phaseolicola growing at 18 °C. The signal is processed and transduced to some effector molecule that will act upon the repressor of phaseolotoxin genes. At 28 °C, the genes involved in the synthesis of phaseolotoxin, such as amtA, are negatively regulated by a repressor molecule that may bind to TRR motifs; when the temperature is lowered to 16 to 18 °C, the signal is processed and relieves repression mediated by the repressor. These genes are then actively expressed [57]. Analysis of the reverse transcription of phtL, the intergenic region of phtMN and amtA, confirmed that expression of these genes in enhanced by components present in leaf extracts. Additionally, phtB and desI, which belong to the phtA and phtD operons, respectively, showed a 1.5-fold increase in expression, values that are statistically significant on the basis of microarray analysis [58]. Recent studies have revealed that the two-component system GacA/GacS controls production of phaseolotoxin. In fact, gacA− and gacS− mutants in P. syringae pv. phaseolicola do not produce phaseolotoxin in vitro [59]. These results contradict previous reports, in which a gacS (lemA) mutant was observed that could produce phaseolotoxin [60]; however, the latter results are consistent with numerous data demonstrating the critical role of the highly conserved GacA/GacS system in achieving full virulence and pathogenicity in other pathovars of P. syringae [59].
4. Mangotoxin
Mangotoxin is a recently discovered antimetabolite toxin produced mainly by P. syringae pv. syringae. Until now, few other mangotoxin-producing pathovars had been detected, although it would be necessary to check a large number of strains belonging to different pathovars to be conclusive [61]. So far, mangotoxin production has been detected in two strains of P. syringae, pv. avellanea and pv. pisi [21]. Mangotoxin seems to be an oligopeptide [61], and preliminary data suggest that it is composed by of two amino acids linked to a sugar residue (unpublished data). Mangotoxin inhibits ornithine acetyl-transferase (OAT), with inhibitory activity comparable to that produced by the chemical inhibitor p-chloromercuribenzoic acid (PCMB) [61]. Experiments to determine the contribution of mangotoxin production to the severity of disease symptoms and epiphytic fitness were performed using wild-type, toxin-producing strains as well as mutants that are mangotoxin-defective [3]. These experiments revealed that the necrotic symptoms produced by wild-type P. syringae pv. syringae UMAF0158 (CECT7752) were significantly more extensive and frequent than those produced by mangotoxin-defective mutants, indicating that mangotoxin-defective mutants are less virulent. Therefore, mangotoxin was considered a virulence factor [3,61]. Moreover, similar population densities of mangotoxin-producing strains and defective mutants were detected when hosts were inoculated individually. However, when the wild type UMAF0158 was co-inoculated with a mangotoxin-defective mutant, the mutant had lower population densities. These results suggested that P. syringae pv. syringae strains producing mangotoxin were more able to colonize the phyllosphere than non-producing strains, even in direct competition [3].
There is still little information available about mangotoxin. Experiments to identify the genes involved in the production of mangotoxin are ongoing. The information that is available comes mainly from analyses of mangotoxin-defective mutants by integration of the miniTn5 transposon. The sequence comparison of disrupted genes from several miniTn5 mutants showed that, as was observed with both tabtoxin and phaseolotoxin, the global regulator system gacA/gacS maybe also be involved in the regulation of the mangotoxin production [62]. This regulatory system is composed of two essential genes, gacA and gacS, encoding a sensor kinase and response regulator, respectively, which control the production of multiple secondary metabolites and are directly related to pathogenesis [63].
Other genes involved in mangotoxin production have been detected (Figure 6, Table 3), including mgoA [62]. The mgoA gene showed high similarity to non-ribosomal peptide synthetase (NRPS), an enzymatic system involved in the synthesis of antibiotic peptides via the non-ribosomal thiotemplate mechanism of biosynthesis. Analysis of the predicted amino acid sequence from mgoA indicates that it contains only one amino acid activation module typical of a functional NRPS. It contains the typical aminoacyl adenylation domain, found at the N-terminal end, which seems to be responsible for recognizing and adenylating of the carboxylic acid of the amino acid substrate [64]. A condensation domain is conventionally fused to the amino-terminal end of modules accepting acyl groups from the preceding module [65]. Three potential reductase domains were also identified at the carboxyl-terminal end of MgoA [62]. The gene mgoA is surrounded by several genes (Figure 6, Table 3), whose functions are still unknown. The genes mgoB, mgoC and mgoD, corresponding to the ORFs 3, 4 and 6, allow a hypothetical protein group, whose putative function is very difficult to establish because it does not have a clear functional domain. However, a very recent study undertaken to characterize the transcriptional organization of the mgo gene cluster has determined the possible functions of mgoB, mgoC and mgoD [66]. In this study, Arrebola et al. [66] propose that both mgoB and mgoC could be oxygenases. Specifically, mgoB's product is similar to the Haem oxygenase-like, multi-helical superfamily, and mgoC bears significant similarity to the p-aminobenzoate N-oxygenase of Streptomyces thioluteus. In contrast, mgoD is similar to two possible domains, a polyketide cyclase and a lipid transporter domain. These four genes, mgoBCAD, are co-transcribed in only one transcript, the absence of which produces mangotoxin-defective mutants. Upstream of mgoB is located a functional promoter, where oligonucleotides related to a known transcription factors rpoD (σ70) binding site are present. A functional terminator has been located downstream of mgoD as well, characterizing the complete mgo operon [66]. This operon is highly homologous to the pvf gene cluster identified in P. entomophila, a bacterium that is lethal to Drosophila melanogaster [67]. Vallet-Gely et al. [67] have proposed that the pvf gene cluster of P. entomophila could perform a regulatory role involved in the production of virulence factors in Pseudomonas species [67]. Based on studies conducted by Vallet-Gely et al., a possible regulatory role for the mgo operon could also be proposed. Performing the same experiment described by Vallet-Gely et al., Arrebola et al. observed a complementation when extract from wild type UMAF0158 was used in cultures containing mutants for the gac and mgo operons, which led them to suggest a possible regulatory role for the mgo operon similar to that proposed by Vallet-Gely et al. for the pvf genes [66].
|NCBI Protein id
|Putative function
|Ortholog genes a
|Ps. syringae B728a
|Ps. tomato
|Ps. phaseolicola
|aldo/keto reductase
|regulatory protein GntR
|ORF3 (mgoB)
|Hypothetical protein
|ORF4 (mgoC)
|Hypothetical protein
|ORF5 (mgoA)
|non-ribosomal peptide synthetase
|ORF6 (mgoD)
|Hypothetical protein
aGenes annotated in NBCI data base with 100% of coverage and identity with corresponding gene in Ps pv. syringae UMAF0158 (CECT7752). Accession numbers of the P. syringae strains used in the preparation of this table are as follow: Ps syringae B728a (CP000075), Ps tomato DC3000 (AE16853.1) Ps phaseolicola 1448A (NC_005773).
At the same time, these authors also obtained evidence of other genes, absent in the genome of P. syringae pv. syringae B728a, which could also be involved in specific processes for the production of mangotoxin, suggesting specific reactions for mangotoxin production encoded out of the predicted mgo operon containing the putative NRPS of mgoA [62]. These specific genes were located in the genome of the wild type P. syringae pv. syringae UMAF0158 and present an operon-like structure (unpublished data). This cluster consists of six ORFs that have been given the acronym mbo, for mangotoxin biosynthetic operon, and each ORF has been ordered from A to F. The mutation of this cluster results in mangotoxin-defective mutants, and the most surprising result is that when theses mbo genes are introduced into P. syringae pv. syringae B728a on a plasmid, this non-producing strain turns into a mangotoxin-producing strain [67]. For this reason, Carrión et al. [67] have attributed this biosynthetic capacity to the mbo operon. The specificity of these genes makes it possible to develop a PCR-based detection method for mangotoxin producing strains [67], providing easy identification for these kind of strains in field samples (unpublished data).
5. Conclusions
After compilation of the information discussed in this review, it is possible to conclude that Pseudomonas syringae is a bacterial species that is perfectly adapted to the plant environment, and an important part of this adaptation is the production of wide range of virulence factors. The antimetabolite toxins are a group of small, peptidic molecules composed of a few amino acids linked to another chemical structure, which can act as inhibitors of key enzymes in the nitrogen metabolism of the plant host. These toxins seem not to be exclusive to any particular pathovar or plant host, but neither do they seem to be produced by other phytopathogenic species. From the genetic point of view, the biosynthesis of this kind of toxin is related to non-ribosomal synthesis, where the biosynthetic genes are grouped in a gene cluster, and all the enzymes required for the assembly and maturation of the toxin, even those needed for secretion of it, are located together [43]. These clusters can be located in genomic islands that can be transferred horizontally between strains of different pathovars [55]. This is in accordance with the non-specific production of toxins in the P. syringae pathovars. All of the genomic islands can be regulated by the gacA/gacS system, which is also in accordance with the hypothesis of horizontal transfer, and the regulation of antimetabolite toxin production has to be accomplished by a common regulation system to prevent excessive production. Finally, the presence of genes exclusive to the antimetabolite toxins have allowed researchers to develop molecular techniques to detect toxin-producing strains related with important diseases, making possible the early detection and control of diseases.
The realization of this manuscript has been supported by funding from Consejería de Innovación, Ciencia y Empresa, Secretaría General de Universidades, Investigación y Tecnología, Junta de Andalucía, Spain (Proyecto de Excelencia P07-AGR-2471).
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What chicken are you picking?
When it comes to fried chicken, New Orleans knows how to get down right delicious. Here's a list of nine spots that will surely tease your taste buds.
Willie Mae's Scotch House
The famous, spicy-battered fried chicken at this New Orleans comfort food staple keeps crowds coming back. Even President Obama stopped by during his Crescent City visit to savor the flavor. Located in the Treme neighborhood, these crispy, juicy pieces of goodness gained nods from food writers as "America's Best Fried Chicken" and even a James Beard Award. Arrive early to avoid the lunch rush. Plus, don't forget the sides! Try your hand at the red beans and rice, mac and cheese or the fried okra. (Photo courtesy: Willie Mae's)
Dooky Chase
Iconic Creole chef Leah Chase still rocks the kitchen in her 90's, and New Orleans food writers sing praises about her fried chicken. She can now even boast a James Beard Lifetime Achievement Award. From famous fans to regular customers, the legendary Leah Chase captures hearts and stomachs with her down-home cooking.
Raising Cane's
Do we even need to get in-depth for this one? Since its launch in Baton Rouge, Raising Cane's has grown a household name, and the sauce is the thing of legends. Now, Nation's Restaurant News named the chicken finger masters the fastest-growing chain in the nation for 2017. Check them out at any of their numerous locations across the city. Photo courtesy: Raising Cane's
The crunchy-battered chicken pulled from the fryers at this fast-food giant doesn't hold back the grease or the Cajun flavor. That's probably what makes Popeyes a drive-thru dominating favorite. The chain started as the brainchild of founding restaurateur Al Copeland in 1972. Spicy or mild chicken paired with buttermilk biscuits and New Orleans-style sides makes for a stomach-satisfying meal.
Manchu Food Store
These crispy, juicy, Chinese-spice tinged chicken wings make Manchu Food Store another New Orleans favorite. Many locals flock to this takeout food spot as their go-to for parties or tailgates. If you can brave the bulletproof glass service window and long lines (mostly the long lines), then prepare to get hooked to the fried chicken plates pushed out with super fast service.
Chicken with a side of Southern Hospitality — the many good reviews of Jacques-Imo's focus on the inspired menu AND the fried chicken. Satisfy your chicken craving with Austin Leslie's Fried Chicken, and while you're here, try the shrimp and alligator sausage cheesecake if you have any room left over.
McHardy's Chicken & Fixin'
Hot, fresh, peppery chicken from this takeout spot gives your stomach tasty eats at very reasonable prices. McHardy's is a local favorite. Get your chicken fix along with soul food sides like dirty rice, potato salad and red beans and rice.
Brother's Food Mart
Ok, so don't judge us for this one. We dare you not to have a fried chicken craving after leaving a Brother's Food Mart. Feast on fried chicken for only a few bucks. Plus, the late-night hours and multiple locations make this spot even more of a convenience. Photo courtesy: Brother's Food Mart via Facebook
Coop's Place
This laid-back French Quarter bar and restaurant nails it with their "Cajun Fried Chicken" made with their own bayou blend of seasonings. The entree comes with rabbit and sausage jambalaya and coleslaw. Photo courtesy: Coop's Place

subset: commoncrawl


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Standard Furniture - Search Results/
Ok, quick, name a movie with Kevin Kline, Joan Allen, Sigourney Weaver, Tobey MacGuire, Christina Ricci, and Elijah Wood. I hadn't heard of this movie before I saw it on TV, and I was like, "Wait... wow, look at all these people in this movie. How have I not heard of this before?"
To be honest, I don't know how good of a movie this is... but it just has so many people in it that I really like to watch. I like Kevin Kline a lot because I tend to like his characters. He's great at playing a guy with good intentions that gets himself into some not so ethical situations, but yet, it always seems to rise above it. While Keven Spacey did an enormous performance in American Beauty, I think Keven Kline could have also played that role as well.
And Sigourney Weaver... well, Sigourney and I have a connection, because we literally bumped into each other one time... like actual bumping. I was going to pick up my high school girlfriend at Sacred Heart on 91st and 5th, and when I was turning the corner on 91st and Madison, we walked right into each other. I didn't realize who it was at first, but when I was like "Oh... um.. wow." She smiled and we both walked off to return to our normal lives, forever altered by that single moment. I'm sure she feels different for having met me even to this day.
The movie takes place in the early 70's in suburban Connecticut, with charactors basically stumbling through the search for themselves by doing all the things they shouldn't, with sometimes tragic results. Its sad, sometimes interesting and often somewhat uncomfortable, but it is in these moments of uncomfortable sexuality where the A cast really shines. (Although, since I already said that Christina Ricci was in it, did I even have to mention that there would be some uncomfortable moments like that in it? She was going 17 going on 36 at the time.) No car chases, explosions, special effects... just a really great movie with great actors and some interesting situations.


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Despite historic data, scientific evidence, and common sense, some evolutionists insist that dinosaurs evolved into birds. If you study on it a mite, you'll realize that the idea of something as complex as feathers evolving from scales, the evolution of flying ability, the claim that bird-hipped dinosaurs are not the ones to evolve into birds instead of lizard-hipped, complete rearrangement of internal organs — nope, that dog don't hunt. Add to this an examination of a T. rex brain cast compared to the brains of birds, you'll see that more and more evidence shows that birds and dinosaurs were created separately, and it makes no sense to believe in dinosaur-to-bird evolution.
Evolutionists insist that dinosaurs evolved into birds, despite the strong evidence against it.1 One of the portrayed misconceptions concerns the brains of large predatory dinosaurs like Tyrannosaurus rex that have been depicted as similar to the brains of their bird "descendants." However, CT scans of T. rex skulls give scientists additional details of its brain cavity, demonstrating its large olfactory lobe (for smell) and an overall shape that is much more similar to modern alligators than birds.To read the rest, fly on over to "Tyrannosaurus rex Was No Birdbrain".
Bird brains have a completely different shape from those of dinosaurs and reptiles, with a larger section for processing data. Birds have to do more than sense a food source; they have to be able to discern one food source from another. Alligators merely smell something and snap at it without thinking. Not only is a bird's brain shaped differently, but pound for pound relative to body weight, the typical bird brain is much larger than a typical reptile brain by nearly an order of magnitude (or ten times).


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From race riots to experiences of African-American athletes, Indiana State University students will be presenting research into race relations from noon to 3 p.m. on Dec. 10 in the Cunningham Memorial Library's Events Area.
Students in the history 300 class have spent the semester investigating topics related to protests and race riots that led to the establishment of Indiana State's African and African-American Studies program and the university's African-American Cultural Center.
When Anne Foster, associate professor of history, teaches that 300-level methodology class she likes to connect it to local history. With the studies program celebrating its anniversary, she found a natural fit.
"It helps students connect to the community and to appreciate the rich and interesting history of Terre Haute," she said. "They are usually surprised to learn how much has gone on here."
A sit-in, a race riot and other efforts led to the establishment of an African-American Studies program, which the Indiana Commission on Higher Education approved ISU's in August 1972. ISU's interdisciplinary program is one of the first in the Midwest and one of the earliest in the nation.
"This semester the study of how ISU became an early leader in African and African-American Studies has helped students become more aware of the important role students played in the creating the curriculum," Foster said. "They have also learned a lot about how ISU was a pioneer in promoting integration and education for all people. But it wasn't always easy to be out in front, and it is good for students to learn about that too."
Students' projects include research into the May 1970 race riot, newspaper coverage of that event, general explorations of black and white student relations, experiences of African-American athletes at ISU as well as race relations and civil rights in Terre Haute.
"The students have learned how much work it is to do historical research, but also how exciting it is to find original accounts by participants, and to learn things that no one else knows," Foster said. "One group told me they were cheering in front the microfilm reader one evening when they found the information they were looking for. Another student, an African and African-American Studies major, told me how inspired she has been to learn more about the risks people took to create the major she is now pursuing. All of them, I think, have become passionate about preserving the stories of ISU."
Contact: Anne Foster, Indiana State University, associate professor of history, at 812-237-8432 or firstname.lastname@example.com
Writer: Jennifer Sicking, Indiana State University, associate director of media relations, at 812-237-7972 or email@example.com
From race riots to experiences of African-American athletes, Indiana State University students will be presenting research into race relations from noon to 3 p.m. on Dec. 10 in the Cunningham Memorial Library's Events Area.
'The Miraculous Journey of Edward Tulane' opens July 5
A modern 'Romeo and Juliet' opens June 28
'Ring of Fire' Johnny Cash tribute opens June 21
Indiana State University announces the spring 2019 dean's list
Sycamores keep momentum going in classroom during 2018-19 academic year


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Fire safety recommendations for home care providers
The Department of Social Services has released a number of recommendations for home care providers to help promote basic fire safety to their care recipients. These recommendations include fire safety training for aged care workers and the fitting of additional smoke alarms for care recipients considered to be at greater risk of fire danger.
Read more >
Tags: aged care, government, health, home care, safety, training


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For the Love of Fish
For the Love of Fish
A 59-page perfect bound chapbook of stories and poems about the love/hate dynamics of working in commercial fishing. As part of the Anchored in Deep Water Fisher Poets' Anthology collection.
Add To Cart
The FisherPoets Anthology: Anchored in Deep Water, is a seven-book set of work by writers, poets and songwriters who have performed at the annual FisherPoets Gathering in Astoria, Oregon, edited by fisherpoet Patrick Dixon. Over 40 writers from the USA, Canada and Japan are represented, with seven interviews of the fishermen and women engaged in commercial fishing along the coasts of the North American continent.


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Is instant messaging a valuable collaboration tool or an invasive time-sucker?
It can be both, say researchers from Ohio State University and the University of California. According to The INQUIRER, the researchers found that IM was a distraction for workers when it was part of a communications cocktail including the phone, e-mail and face-to-face conversations. However, it boosted productivity when it was used as a substitute for other types of communications.
Kelly Garrett, an assistant professor of communication at Ohio State who co-authored the study, says many folks use IM in lieu of longer conversations when they need a short answer to a question. They can also quickly check in with co-workers to see if they are available for a confab.
This finding meshes pretty well with another study I wrote about last month. Researchers Sinan Aral, Erik Brynjolfsson and Marshall Van Alstyne found that too much multi-tasking lengthens work projects. I also mentioned the Burton Group's contention that companies should work on creating an optional mix of "attentional" and "attention-shielding" technologies to maximize employee productivity.
A technology like IM puts the productivity ball directly in an employee's court. That's pretty much been my experience and the experience of others here at IT Business Edge as well, judging from a blog post by our Ken-Hardin. Realizing that IM was dominating his work day, Ken halved his messaging volume simply by putting a little more thought into how he used IM. He wrote:
Like in most cases, the underlying problem with this tech was the person using it.


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Shipping reptiles is a very common process that is quite safe when done correctly. We take great care to make sure your snake will arrive to you safely.
The Process
We break shipping up into four phases, each of which is listed below.
After completing your purchase we will reach out to you via telephone or email to begin coordinating shipping. All of our shipments are sent Priority Overnight via FedEx and we typically only ship Monday through Wednesday. We will coordinate the following things with you:
The best day for you to receive the shipment. This needs to be a day where you (or someone 18+ years old) can plan to be home all day to receive and immediately the box. Note: We can ship to commercial addresses, too. Please prepare accordingly if having an animal delivered to your work. If you ask us to ship to your work we are assuming that you cleared this with your employer.
The weather. In the winter it can be too cold and in the summer it can be too hot. Appropriate care must be taken with regards to weather. If it is too hot or too cold we will have to delay shipment. In the winter and summer months this can cause a significant delay between when you purchase your snake and when it can be safely shipped. We can ship safely ship in the mid-30's but do not ship if it gets colder than that. But don't worry about the snake getting too cold; all of our shipments are made using insulated and heated reptile shipping boxes. If it is over 90F, we don't ship. Bottom line is that patience and safety are more important than a speedy arrival. We will ship just as soon as it is safe to do so and we will communicate with you throughout the process.
Why only Monday through Wednesday?
All shipments require a signature for delivery. FedEx cannot leave the box at the door. If the delivery is missed FedEx will re-attempt on the next delivery day. If delivery is missed on a Friday, the next delivery day is Monday. With a live animal shipment, this must be avoided.
Thursday shipping (Friday delivery) is an option but we try to avoid it. Coordinate with us if Friday is the only day that works for you. We can make it happen.
Weekend delivery is not available.
On the actual day of shipping we will do several things:
- We will double-check the weather to be sure nothing has changed.
- We will send you an email re-confirming that you are ready for the shipment.
- We will generate a FedEx shipping label and tracking number. FedEx's automated system will forward you an email with the tracking number.
You will receive two emails on the day of shipping. The first is titled "FedEx Shipment Notification". This is the initial notification that is generated just after we create the shipment using FedEx's on-line system. At this point, the box is still with ECRB. This email contains a 12-digit tracking number that can be used to follow the package as it moves through FedEx's system. The second email you receive from FedEx will be titled, "Tendered to FedEx". This email notifies you that the box has been dropped off at FedEx here in Virginia Beach. You will typically receive this email around 6:00-7:30PM EST. This email will also have a link with the tracking number. From this point on, everything is in the hands of FedEx. [bgarea]
Where Does the Weather Matter?
- We operate out of Southeastern Virginia. The weather here matters the least. This is because we almost always take boxes directly to the FedEx hub at our airport. We do this as late in the day as possible, usually around 7:00PM EST. The box is usually on a plane within an hour or so. This means that the weather here in Virginia Beach is not a significant factor.
- Overnight the box will pass through either Indianapolis, IN or Memphis, TN. These are FedEx's main hubs and all overnight packages pass through one of these locations. We will know in advance (99% of the time) which hub will be used so we can check the weather forecast in these cities to determine if it is safe to ship. The box will spend the majority of the time at the hub so this is the place where the weather matters most. Note that temperature is not the only factor. Memphis often has violent thunderstorms and these storms can also impact flight schedules. It is possible that severe thunderstorm forecasts in Memphis can cause us to delay shipment.
- The weather where you live is the second most important factor. Click here to check the forecast for your location. Depending on how long the box will be on the truck for delivery, the weather in your city can play a major factor in safe shipping.
Using your tracking number and the Internet you can track your package overnight and into the morning. If you are like most people you will track it about a thousand times.
This is the hard part. I recommend going to sleep. Clicking refresh on your browser every 2 minutes will not make the plane fly any faster. 🙂
Early in the morning you should should see that the tracking status of your shipment has changed to "Out for Delivery". It's getting exciting now!
Unfortunately, FedEx cannot guarantee an exact arrival time but they typically provide an estimated "delivery by" time. This is usually before noon on the day of delivery but people who live in more rural or geographically dispersed areas may find that their delivery times are even later than that. All you can do at this point is patiently wait. Do not. Please, please, please. Do not decide to 'run to the store real quick' in the middle of the morning. Across the years I have found that FedEx is guaranteed to attempt delivery in the 15-minute window you just happened to be out at the convenience store. Once the box arrives please open it right away. Please don't forget to send me an email, a text or a phone call to let me now your new snake is safely with you.
Delivery Exceptions
If you receive an email on delivery day with the title "FedEx Delivery Exception" you need to immediately contact FedEx (800-GO-FEDEX) and/or us. We will be trying to contact you via phone, text and email, too. The number one reason for a delivery exception is that FedEx attempted delivery but nobody was home. If this happens you need to immediately call FedEx and nicely explain that your shipment contains a live animal and ask them to please contact the local office and request that the driver come back and re-attempt delivery. If the driver cannot come back you will need to ask the FedEx rep where the box will be at the end of the day so you can drive there in the late afternoon/early evening and pick it up. It is important to avoid having the animal spend an extra night in the box, especially during extreme temperatures.
Can We Speed Up the Process?
Yes! If you would like to avoid the extra wait while your box is "Out for Delivery" you can choose to have the box held for pickup at your local FedEx distribution center. This typically means you can pick the box up at 9AM (your time) rather then waiting for it at home. This is our favorite option because it all but eliminates the weather as a factor on your end. All you need to pick the box up is the tracking number and your picture ID. You can even decide this after the box is with FedEx. If the box is supposed to come to your address but you decide overnight that you want to pick it up you can use the tracking link and change the status of the package to "Hold for Pickup". The on-line system will provide you with the address where the box can be picked up at 9AM. [/bgarea] If you have additional questions about the shipping process, please contact us.


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Handmade In raleigh,NC
The 510 is a small sized archtop. Its aesthetics pay homage to the big guitars of the past, but the 510 is decidedly modern. The 14" lower bout and thinline body make it comfortable for those all night jams. The carved top and back plate can be coupled with a center block for high volume electric playing. If an acoustic sound is more your style the 510 can be built fully hollow with an X-brace top. It is available with standard or frets fanned frets. The 510 is a small modern archtop for a plugged in world.


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London's Anniversary Games in pictures
The best images from London's Anniversary Games as some of the world's top athletes return to the Olympic Stadium one year on from London 2012.
Anticipation built on Friday as London's Olympic Stadium prepared to stage the Anniversary Games, marking one year since the opening of the 2012 Olympics.
Athletes warmed up in the stadium ahead of event on Thursday evening - American hurdler David Oliver among them.
On Friday evening, Jamaican sprinter Usain Bolt arrived at the stadium in Stratford - where he won 100m and 200m gold a year ago - in typically flamboyant style.
Londoner Perri Shakes-Drayton just missed out on the 2012 Games, but on Friday she clocked up a personal best in the 400m hurdles - in some eye-catching footwear.
The Olympic champion and world number one pole-vaulter, America's Jennifer Suhr, was among the competitors. Here she grabs chalk in preparation.
German pole-vaulter Silke Spiegelburg was among those showing off her skills as dusk began to fall over London.
Great Britain's 4x100m women's relay team did not make the 2012 final a year ago, but on Friday night Ashleigh Nelson (pictured), Dina Asher-Smith, Anyika Onuora and Annabelle Lewis won convincingly.
Usain Bolt rounded off Friday's action by powering to a comfortable victory in the men's 100m, recording his fastest time of the year at 9.85 seconds.
Fans had not forgotten Mo Farah's unique "Mobot" celebration from one year ago as the Anniversary Games continued in glorious sunshine on Saturday.
The man himself gave a sell-out crowd plenty to cheer as he eased to victory in the 3,000m, setting a personal best time.
And his fans weren't the only ones celebrating.
Another of Britain's 2012 heroes, Jessica Ennis-Hill, struggled with injury as she came last in the long-jump and ran half-a-second slower than her Olympic time in the hurdles. She said she did not know if she would be fit for the World Championships in August.


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Indies And The Underground
"Exposing New Music To The Blog-Reading Masses"
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Strip Steve's debut Micro Mega, described by its creator as a "psych noir disco p0p trip" came out this week. If that description intrigues you (and/or you're apprehensive about throwing down your cash on just any old pop -meets-disco mashup) listen to a stream of the entire album after the jump.
BNR081: STRIP STEVE – MICRO MEGA by Boysnoize Records


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On Sunday, April 23, the Dallas community came together at Temple Emanu-El to remember the six million Jews murdered during the Holocaust and to honor those who survived.
The theme of Yom Hashoah 2017 was Jewish resistance. On Holocaust Remembrance Day, music, testimonies, and prayers were used to reinforce the message that Jewish resistance in the Holocaust must be remembered and honored in today's time.
"We are here tonight in a society stricken with amnesia to remember," began Rabbi David Stern, addressing a crowd of over 500 individuals in the Stern Chapel. "We are here tonight in a world which too often turns a deaf ear to the cry of the oppressed to listen. We are here in a culture which makes it easy to harden our hearts to the suffering of others, to open our hearts to their struggles. We are here to affirm that 'never again' means 'never again' to us, 'never again' to anyone."
The powerful words "Never Again" are a reminder to all that the tragedy of the Holocaust must not happen again. However, these words have recently lost some of their effects as evidenced by the rise in hate speech and antisemitism across the nation, including Texas. When being a bystander to hatred seems like an easy path, we must stand up and remember those who resisted and fought for others in the face of unspeakable horrors and death.
Holocaust survivors sat in the first rows at the commemoration. Their strength and unrelenting faith serve as evidence to what humans can endure and overcome with enough hope and courage.
"Resistance of any kind during the Holocaust required great courage," said Museum President and CEO Mary Pat Higgins. "Today we remember and marvel at Jewish resistance during the Holocaust, the armed resistance in ghettos and death camps across Poland most notably the Warsaw Ghetto Uprising and the Sonderkommando uprisings in the death camps of Auschwitz-Birkenau, Treblinka and Sobibor."
The Jews had everything taken away from them by the Nazis and their oppressors. Without family, food, weapons, or freedom, many refused to be completely dehumanized and stood up for themselves and others by participating in resistance activities.
To further speak on the theme of Jewish resistance, the testimonies of three Holocaust survivors who fought against Nazi oppression were read aloud by their respective children.
Mark Jacobs read the testimony of his father Mike Jacobs; Julie Meetal Berman read the testimony of her father Les Mittelman; Marsha Gaswirth read the testimony of her father Leon Bakst.
Following each testimony, a member of the Israeli Scouts joined a speaker to light two of the six white candles used to signify the six million Jews murdered in the Holocaust.
The Honorable Florence Donald Shapiro, the Museum's Chairman of the Board, ended the evening with a powerful message to all in attendance.
"The education the school students receive in the Museum, and how we honor our survivors, their lost loved ones, and the victims of the Holocaust, is through these stories, through this education. This is how we honor the children of the Holocaust: by educating the children of today. In the year 2017, this is how we continue our resistance."
– Janet Montealvo Hitt, Marketing Coordinator, Dallas Holocaust Museum/Center for Education and Tolerance


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In a recent meta-analysis, researchers analyzed the results of 29 trials and 17,922 patients suffering from chronic pain. Conditions included: neck pain, shoulder pain, low-back pain, headaches & migraines, and osteoarthritis of the knee.
From the original data set, 20 trials and 6,736 patients were used for the final conclusions, due to the fact that they included long-term follow-up data, which ultimately demonstrated that after a course of treatment, the effects of acupuncture treatments persist over a 12 month period.
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June 2019
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From today's New York Times:
The most significant harbinger from the Ohio race may not be the narrow margin, but the turnout gap between the most and least heavily populated parts of a district that absorbs the close-in suburbs of Columbus and rural stretches of central Ohio.
In both Franklin County, which includes Columbus, and Delaware County, the fast-growing suburb just north of Ohio's capital, 42 percent of voters turned out. But in the five more lightly populated counties that round out the district, turnout ranged from 27 to 32 percent.
This is an ominous sign for Republicans: The highest-income and best-educated elements of the electorate — those deeply uneasy with President Trump — are showing the most interest in voting. Defending a few dozen districts that are either more heavily urban or feature a similar demographic mix as Ohio's 12th District, Republicans will need to find a way to win back suburbanites or better galvanize rural voters. If they do not, their House majority will slip away.
OK, let's reread that last sentence. To win, Republicans EITHER have to win back the country club set OR they have they have to ratchet up their appeal to the unwashed.
As Greg Sargent observes, the Republican establishment has made its choice. They have well and truly bitten the bullet. They have decided which horse to ride on:
The key to this is that Trump and Republicans have been doing everything they can to energize those rural Trump-leaning areas. If you look at the ads that Republicans ran against O'Connor — see here and here — they amount to a festival of Nancy Pelosi-bashing and relentless culture-war demagoguing about crime, the menacing immigrant invasion, and the alleged threat that "the Resistance" poses to traditional values. With the GOP tax cut mostly disappearing from GOP messaging, this represents a last-ditch effort to galvanize Trump's rural base.
It's working. But it's not working well enough. Yesterday, 42 percent of the voters in suburban Franklin County showed up, compared with between 27 to 32 percent in the twelfth district's five most rural counties.
In the rural areas, the subset of folks who chose to vote were even trumpier than they were in 2016. But lots and lots of the country club set are gone, gone, gone.
What all this tells me is that Trump and his enablers are energizing A PORTION of the most ignorant and gullible of the rural white vote. "Trump's base" is being winnowed down to a crazy hard core.
It is a loser's strategy. Winning politicians practice the art of addition. Only a loser practices the politics of subtraction.
We were told long ago that Trump's goal was to lose the 2016 election and then monetize his increased fame. Having failed in that goal two years ago, he persists.


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Know an amateur mixologist? Check out these fab gifts for their home bar.
-PJ Gach
It's getting down to crunch time! Still working on your gift guide and feeling a little stressed? Take a look at these fabulous treats for the wine lover or cocktail aficionado on your list. The best thing about these gifts—well there are two: (1) These gifts last a long time and (2) You can find them at fine wine shop or liquor stores nationwide.
The Clicquot Fridge ($54.99), designed with a retro nod to 50's refridgerators, this fun champagne fridge will keep a bottle of champagn cool up to two hours. Perfect for spring and summer picnics.
Naturally Clicquot ($49.99). Internationally known interior designer, Cedric Ragot, this eco-friendly case will keep bubbly chilled for up to two hours. Best of all, it's made from of 100% biodegradable potato and starch paper.
Gekkeikan Black & Gold Gift Set ($14.99). Looking for a unique sake for a unique person? Black & Gold blends two sakes, with tropical fruit aromas, papaya taste and a savory finish. The gift set comes wth two cold sake glasses. Black & Gold is sulfite and gluten-free.
The Manly Man Holiday Gift Guide
SIMI is Sonoma Taster by Out of the Box Collective ($$75.00 with the 2010 SIMI Landslide Cabernet Sauvignon, $65 with the 2011 SIMI Russian River Valley Reserve Chardonnay--online and at the SIMI Winery Tasting Room). Inside this eco-friendly tote is a whole bunch of yummies! SIMI Executive Chef Kolin Vazzoler and Out of the Box Collective Founder, Jennifer Piette selected goods from their favorite Sonoma County purveyors like Merchants and Millers, Black Pig Meat Co., WholeVine Products, Girl and the Fig, and a custom SIMI Sonoma County Chardonnay infused jam from Dry Creek Peach and Produce. Pick either the 2011 SIMIRussianRiverValley Reserve Chardonnay or the 2010 SIMI Landslide Cabernet Sauvignon.
Inniskillin Vidal Icewine ($40). Vidal is a hybrid-A mix of Ugni Blanc and Seibel, it's harvested late in the season in Ontario. It's a delicious soft, fruity wine that bring the flavors of summer (peaches, apricots) with yummy jammy undertones. It's perfect with anything.
American Harvest Stemless Martini Glass Pack ($23.99). You could go all Mad Men or James Bond with this stemless Martini gift pack that contains 1 8oz glass and a 750 ml bottle of American Harvest vodka. But who hasn't wanted to unwind at the end of the day with a perfectly balanced Martini? Especially if it's made from organic vodka!
Bärenjäger Honey & Bourbon Rocks Glass Pack ($28.99). I've recently gotten into bourbon and it's such a soothing libation! Know someone who loves "sippin' whiskey," they'll really savor the Bärenjäger Honey & Bourbon set. You'll find a 750ml bottle and an 9oz rocks glass etched with the Bärenjäger logo.


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Mia is waiting impatiently for her partner, to join her in bed... Her moans of pleasure prove how excited she gets, when the guy stuffs his dick in her naughty pussy. She was wrong to imagine that it couldn't get any better, as the atmosphere intensifies, when she gets banged in her tight ass. See details!
- amadeus
- : 6min 56sec
- : 16 May 2015
- Exclusive video


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90sFest Brings the 90s Back to Columbus, OH
Friday, September 16th – 90sFest hit Columbus, OH with performances by Sister Hazel, Bone Thugs N Harmony, Sisqo, Vertical Horizon, DJ Jazzy Jeff, Saved by the 90s and RATM2. The event was packed with nostalgic 90s fun including a 90s Nick obstacle course brought to the event by presenting partner The Splat. For more on the event, check out this great piece by Columbus Alive: http://www.columbusalive.com/content/blogs/thelatest/2016/09/festival-review-90sfest.html


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Illustrated by Emily Turner; Photo: Michelangelo Gratton/Rex USA.
Modern spiritual teachers like Gabrielle Bernstein and Mastin Kipp bring up the other F-word all the time: forgiveness. But, it's an age-old pursuit, and often, self-forgiveness is no easy task. "I think it's absolutely critical to being on a path of wellness and fulfillment," says holistic lifestyle consultant and Zen Buddhist Maggie Lyon. The problem is that forgiving yourself for all of the stupid things you do (yes, you) is much harder than it seems, especially in our do-better culture, where constant pressure to be the best makes us feel like any slip-up will derail our entire livelihood. (Thanks, New York.)
So, why can't you just give yourself a break and move forward?
Lyon says it's because there's too much focus on forgiveness and not enough attention paid to what lies beneath it. "It really starts from this place of deep, no-nonsense, unyielding self-love," she explains, "meaning that no matter what, no matter the suffering, mistakes, and missteps that we make, whatever it is, we treat ourselves with such tenderness that forgiveness is part and parcel of that."
And loving yourself enough to let go doesn't mean you're letting yourself off the hook for bad behavior, Lyon says. You can forgive yourself and also take steps toward not allowing it to happen again. "You can be okay, even though feelings of anger and disappointment and difficulty and harshness are floating around, and you're nailing yourself for whatever you did. Allow it and accept it — you're still working on changing your habits and being better."
Here, Lyon shares a simple visualization exercise to help you get there.
A Simple Forgiveness Visualization
1. Stop and sit. Close your eyes and get in touch with your breath, focusing on your inhales and exhales. "Bring about in your mind's eye what you've done, the act you're so obsessed and crazy about, and really feel all of the bad stuff it creates in the body, and just be with it," Lyons says.
1. Stop and sit. Close your eyes and get in touch with your breath, focusing on your inhales and exhales. "Bring about in your mind's eye what you've done, the act you're so obsessed and crazy about, and really feel all of the bad stuff it creates in the body, and just be with it," Lyons says.
2. Soften your thoughts. As you're picturing the situation, imagine the edges around it softening. "Accept the fact that it's happened, and at the same time start to wrap that act with love, try to just be softer and nicer with it."
3. Imagine yourself as someone you love. Self-love is hard, so use the love you feel for others to begin to imagine what it might feel like. "Call up a being that you love unconditionally, someone who you can easily see as someone deserving of love," Lyon says, like your perfect baby daughter or niece or amazingly generous best friend. Picture yourself as that person, and see if you can begin to apply the love you feel for them to yourself. "Come back to the breath and see if everything releases a little bit, if there's some kind of ease it has created in your body, and then open your eyes."
This post was authored by Lisa Elaine Held.