Expression and crystallization of SeDsbA, SeDsbL and SeSrgA from Salmonella enterica serovar Typhimurium

Pathogens require protein-folding enzymes to produce functional virulence determinants. These foldases include the Dsb family of proteins, which catalyze oxidative folding in bacteria. Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K-12 and these mechanisms have been extrapolated to other organisms. However, recent research indicates that the K-12 complement of Dsb proteins is not common to all bacteria. Importantly, many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence. To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins, Salmonella enterica serovar Typhimurium has been focused on. This Gram-negative bacterium contains three DsbA proteins: SeDsbA, SeDsbL and SeSrgA. Here, the expression, purification, crystallization and preliminary diffraction analysis of these three proteins are reported. SeDsbA, SeDsbL and SeSrgA crystals diffracted to resolution limits of 1.55, 1.57 and 2.6 Å and belonged to space groups P21, P21212 and C2, respectively.

Structural and functional characterization of three DsbA Paralogues from Salmonella enterica Serovar Typhimurium

In prototypic Escherichia coli K-12 the introduction of disulfide bonds into folding proteins is mediated by the Dsb family of enzymes, primarily through the actions of the highly oxidizing protein EcDsbA. Homologues of the Dsb catalysts are found in most bacteria. Interestingly, pathogens have developed distinct Dsb machineries that play a pivotal role in the biogenesis of virulence factors, hence contributing to their pathogenicity. Salmonella enterica serovar (sv.) Typhimurium encodes an extended number of sulfhydryl oxidases, namely SeDsbA, SeDsbL, and SeSrgA. Here we report a comprehensive analysis of the sv. Typhimurium thiol oxidative system through the structural and functional characterization of the three Salmonella DsbA paralogues. The three proteins share low sequence identity, which results in several unique three-dimensional characteristics, principally in areas involved in substrate binding and disulfide catalysis. Furthermore, the Salmonella DsbA-like proteins also have different redox properties. Whereas functional characterization revealed some degree of redundancy, the properties of SeDsbA, SeDsbL, and SeSrgA and their expression pattern in sv. Typhimurium indicate a diverse role for these enzymes in virulence.

A new Salmonella typhimurium NM5004 strain expressing rat glutathione S-transferase 5-5 : use in detection of genotoxicity of dihaloalkanes using an SOS/umu test system

The Escherichia coli mu operon was subcloned into a pKK233-2 vector containing rat glutathione S-transferase (GST) 5-5 cDNA and the plasmid thus obtained was introduced into Salmonella typhimurium TA1535. The newly developed strain S.typhimurium NM5004, was found to have 52-fold greater GST activity than the original umu strain S.typhimurium TA1535/pSK1002. We compared sensitivities of these two tester strains, NM5004 and TA1535/ pSK1002, for induction of umuC gene expression with several dihaloalkanes which are activated or inactivated by GST 5-5 activity. The induction of umuC gene expression by these chemicals was monitored by measuring the cellular P-galactosidase activity produced by umuC'lacZ fusion gene in these two tester strains. Ethylene dibromide, 1-bromo-2-chloroethane, 1,2-dichloroethane, and methylene dichloride induced umuC gene expression more strongly in the NM5004 strain than the original strain, 4-Nitroquinoline 1-oxide and N-methyl-N'-nitro-N-nitrosoguanidine were found to induce umuC gene expression to similar extents in both strains. In the case of 1-nitropyrene and 2-nitrofluorene, however, NM5004 strain showed weaker umuC gene expression responses than the original TA1535/ pSK1002 strain, 1,2-Epoxy-3-(4'-nitrophenoxy)propane, a known substrate for GST 5-5, was found to inhibit umuC induction caused by 1-bromo-2-chloroethane. These results indicate that this new tester NM5004 strain expressing a mammalian GST theta class enzyme may be useful for studies of environmental chemicals proposed to be activated or inactivated by GST activity.

Human glutathione S-transferase T1-1 enhances mutagenicity of 1,2-dibromoethane, dibromomethane and 1,2,3,4-diepoxybutane in Salmonella typhimurium

The rat theta class glutathione S-transferase (GST) 5-5 has been shown to affect the mutagenicity of halogenated alkanes and epoxides. In Salmonella typhimurium TA1535 expressing the rat GST5-5 the number of revertants was increased compared to the control strain by CH2Br2, ethylene dibromide (EDB) and 1,2,3,4-diepoxybutane (BDE); in contrast, mutagenicity of 1,2-epoxy-3-(4'-nitrophenoxy)propane (EPNP) was reduced. S.typhimurium TA1535 cells were transformed with an expression plasmid carrying the cDNA of the human theta ortholog GST1-1 either in sense or antisense orientation, the latter being the control. These transformed bacteria were utilized for mutagenicity assays. Mutagenicity of EDB, BDE, CH2Br2, epibromohydrin and 1,3-dichloroacetone was higher in the S.typhimurium TA1535 expressing GSTT1-1 than in the control strain. The expression of active enzyme did not affect the mutagenicity of 1,2-epoxy-3-butene or propylene oxide, GSTT1-1 expression reduced the mutagenicity of EPNP. Glutathione S-transferase 5-5 and GSTT1-1 modulate genotoxicity of several industrially important chemicals in the same way. Polymorphism of the GSTT1 locus in humans may therefore cause differences in cancer susceptibility between the two phenotypes.

Expression of mammalian glutathione S-transferase 5-5 in Salmonella typhimurium TA1535 leads to base-pair mutations upon exposure to dihalomethanes

Dihalomethanes can produce liver tumors in mice but not in rats, and concern exists about the risk of these compounds to humans. Glutathione (GSH) conjugation of dihalomethanes has been considered to be a critical event in the bioactivation process, and risk assessment is based upon this premise; however, there is little experimental support for this view or information about the basis of genotoxicity. A plasmid vector containing rat GSH S-transferase 5-5 was transfected into the Salmonella typhimurium tester strain TA1535, which then produced active enzyme. The transfected bacteria produced base-pair revertants in the presence of ethylene dihalides or dihalomethanes, in the order CH2Br2 > CH2BrCl > CH2Cl2. However, revertants were not seen when cells were exposed to GSH, CH2Br2, and an amount of purified GSH S-transferase 5-5 (20-fold excess in amount of that expressed within the cells). HCHO, which is an end product of the reaction of GSH with dihalomethanes, also did not produce mutations. S-(1-Acetoxymethyl)GSH was prepared as an analog of the putative S-(1-halomethyl)GSH reactive intermediates. This analog did not produce revertants, consistent with the view that activation of dihalomethanes must occur within the bacteria to cause genetic damage, presenting a model to be considered in studies with mammalian cells. S-(1-Acetoxymethyl)GSH reacted with 2′-deoxyguanosine to yield a major adduct, identified as S-[1-(N2-deoxyguanosinyl)methyl]GSH. Demonstration of the activation of dihalomethanes by this mammalian GSH S-transferase theta class enzyme should be of use in evaluating the risk of these chemicals, particularly in light of reports of the polymorphic expression of a similar activity in humans.

The genetics and structure of the O-specific polysaccharide of Yersinia pseudotuberculosis serotype O:10 and its relationship with Escherichia coli O111 and Salmonella enterica O35

The O-specific polysaccharide (OPS) is a variable constituent of the lipopolysaccharide of Gram-negative bacteria. The polymorphic nature of OPSs within a species is usually first defined serologically, and the current serotyping scheme for Yersinia pseudotuberculosis consists of 21 O serotypes of which 15 have been characterized genetically and structurally. Here, we present the structure and DNA sequence of Y. pseudotuberculosis O:10 OPS. The O unit consists of one residue each of d-galactopyranose, N-acetyl-d-galactosamine (2-amino-2-deoxy-d-galactopyranose) and d-glucopyranose in the backbone, with two colitose (3,6-dideoxy-l-xylo-hexopyranose) side-branch residues. This structure is very similar to that shared by Escherichia coli O111 and Salmonella enterica O35. The gene cluster sequences of these serotypes, however, have only low levels of similarity to that of Y. pseudotuberculosis O:10, although there is significant conservation of gene order. Within Y. pseudotuberculosis, the O10 structure is most closely related to the O:6 and O:7 structures.

A taxonomic assessment of the Australian Dusky Antechinus Complex: A new species, the Tasman Peninsula Dusky Antechinus (Antechinus vandycki sp. nov.) and an elevation to species of the Mainland Dusky Antechinus (Antechinus swainsonii mimetes (Thomas))

In 2014, the northern outlying population of carnivorous marsupial Dusky Antechinus (Antechinus swainsonii) was nominated a new species, A. arktos. Here, we describe a further new species in the dasyurid A. swainsonii complex, which now contains five taxa. We recognise two distinct species from Tasmania, formerly represented by A. swainsonii swainsonii (Waterhouse); one species (and 2 subspecies) from mainland south-eastern Australia, formerly known as A. swainsonii mimetes (Thomas) and A. swainsonii insulanus Davison; and one species from the Tweed Caldera in mid-eastern Australia, formerly known as A. s. mimetes but recently described as A. arktos Baker, Mutton, Hines and Van Dyck. Primacy of discovery dictates the Tasmanian Dusky Antechinus A. swainsonii (Waterhouse) is nominate; the Mainland Dusky Antechinus taxa, one raised from subspecies within A. swainsonii mimetes (Thomas) is elevated to species (now A. mimetes mimetes) and the other, A. swainsonii insulanus Davison is transferred as a subspecies of A. mimetes (now A. mimetes insulanus); a species from Tasmania, the Tasman Peninsula Dusky Antechinus, is named A. vandycki sp. nov. These taxa are strongly differentiated: geographically (in allopatry), morphologically (in coat colour and craniodental features) and genetically (in mtDNA, 7.5-12.5% between species pairs).

Genome sequence of marine bacterium idiomarina sp. strain 28-8, isolated from Korean ark shells

Idiomarina sp. strain 28-8 is an aerobic, Gram-negative, flagellar bacterium isolated from the bodies of ark shells (Scapharca broughtonii) collected from underwater sediments in Gangjin Bay, South Korea. Here, we present the draft genome sequence of Idiomarina sp. 28-8 (2,971,606 bp, with a G+C content of 46.9%), containing 2,795 putative coding sequences.

Validation and reliability of a Spanish version of Simple Shoulder Test (SST-Sp)

Background: The Simple Shoulder Test (SST-Sp) is a widely used outcome measure. Objective: The purpose of this study was to develop and validate a Spanish-version SST (SST-Sp). Methods: A two-stage observational study was conducted. The SST was initially cross-culturally adapted to Spanish through double forward and backward translation and then validated for its psychometric characteristics. Participants (n = 66) with several shoulder disorders completed the SST-Sp, DASH, VAS and SF-12. The full sample was employed to determine factor structure, internal consistency and concurrent criterion validity. Reliability was determined in the first 24–48 h in a subsample of 21 patients. Results: The SST-Sp showed three factors that explained the 56.1 % of variance, and the internal consistency for each factor was α = 0.738, 0.723 and 0.667, and reliability was ICC = 0.687–0.944. The factor structure was three-dimensional and supported construct validity. Criterion validity determined from the relationship between the SST-Sp and DASH was strong (r = −0.73; p < 0.001) and fair for VAS (r = −0.537; p < 0.001). Relationships between SST-Sp and SF-12 were weak for both physical (r = −0.47; p < 0.001) and mental (r = −0.43; p < 0.001) dimensions. Conclusions: The SST-Sp supports the findings of the original English version as being a valid shoulder outcome measure with similar psychometric properties to the original English version.

Complete genome sequence of a novel zantedeschia mild mosaic virus isolate: The first report from Australia and from Alocasia sp

The complete genome of an Australian isolate of zantedeschia mild mosaic virus (ZaMMV) causing mosaic symptoms on Alocasia sp. (designated ZaMMVAU) was cloned and sequenced. The genome comprises 9942 nucleotides (excluding the poly-A tail) and encodes a polyprotein of 3167 amino acids. The sequence is most closely related to a previously reported ZaMMV isolate from Taiwan (ZaMMV-TW), with 82 and 86 % identity at the nucleotide and amino acid level, respectively. Unlike the amino acid sequence of ZaMMV-TW, however, ZaMMV-AU does not contain a polyglutamine stretch at the N-terminus of the coat-protein-coding region upstream of the DAG motif. This is the first report of ZaMMV from Australia and from Alocasia sp.