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Abstract: The complete 98,192bp nucleotide sequence was determined for plasmid pA81, which is harbored by the haloaromatic acid-degrading bacterium Achromobacter xylosoxidans A8. The majority of the 103 open reading frames identified on pA81 could be categorized as either “backbone” genes, genes encoding (halo)aromatic compound degradation, or heavy metal resistance determinants. The backbone genes controlled conjugative transfer, replication and plasmid stability, and were well conserved with other IncP1-β plasmids. Genes encoding (halo)aromatic degradation were clustered within a type I transposon, TnAxI, and included two ring-hydroxylating oxygenases (ortho-halobenzoate oxygenase, salicylate 5-hydroxylase) and a modified ortho-cleavage pathway for chlorocatechol degradation. The cluster of heavy metal resistance determinants was contained within a Type II transposon TnAxII, and included a predicted P-type ATPase and cation diffusion facilitator system. Genes identical to those carried by TnAxI and TnAxII were identified on other biodegradative/resistance plasmids and genomic islands, indicating an evolutionary relationship between these elements. Collectively, these insights further our understanding of how mobile elements, and interactions between mobile elements affect the fate of organic and inorganic toxicants in the environment. [Copyright &y& Elsevier]

Achromobacter xylosoxidans strain A8, isolated from soil contaminated with polychlorinated biphenyls (PCBs), is able to use 2-clorobenzoate (2-CB) and 2,5-dichlorobenzoate (2,5-DCB) as sole sources of carbon and energy. The genome of this strain contains two large conjugative plasmids pA81 and pA82. A cluster of genes homologous to genes of a modified ortho-cleavage pathway was identified on the 12.4 kbp fragment of pA81. The genes, mocpR-ABCD, are highly homologous to the cbnR-ABXCD genes on plasmid pENH91 from Ralstonia eutropha ENH91, the tetR-CDXEF genes from Pseudomonas chlororaphis RW71 and tcbR-CDXEF genes identified on plasmid pP51 from Pseudomonas sp. strain P51. The structures of mocp, cbn, tet and tcb gene clusters are completely conserved in these bacteria. However, the sequences flanking the mocp genes differ from the sequence surrounding the tcb genes. A gene for IS1600 transposase, found on the ends of cbn genes, was identified only downstream from the mocp genes. The vicinity of the transposase gene and the localization of the mocp genes on the conjugative plasmid suggest that chlorocatechol degradation genes are transferable. Hybridization analysis confirmed that mocp genes are located only on pA81, which is thereby essential for the degradation of CBs by this strain. Individual genes were cloned, expressed in Escherichia coli and their activities confirmed by reaction with suitable substrates. [Copyright &y& Elsevier]

The Gram-positive Antarctic bacterium Arthrobacter sp. C2-2 contains two, possibly three cold-active isoenzymes of β-galactosidase. The C2-2-1 isoenzyme was cloned, purified and characterised. This β-galactosidase was classified as being a member of the Family 2 of glycosidases. It is a homotetrameric enzyme, each subunit being composed of 1023 amino acids and it shows great activity towards lactose as a substrate. The C2-2-1 isoenzyme is particularly cold-active, compared to other β-galactosidases including those from some closely-related bacteria, retaining 20% of activity at 10 °C compared with maximum values. The temperature optimum of the purified enzyme was 40 °C using lactose as the substrate. The enzyme is particularly thermolabile, losing all activity within 10 min at 50 °C. The isoelectric point of the enzyme was 5.9. Dithiothreitol and Mg2+ ions were strong activators, whereas Cu2+, Al3+ and Tris were strong inhibitors of activity. The enzyme exhibited transglycosylation ability and the highest concentration of trisaccharides (34 mM) was formed after 10 h at 15 °C, which is an activity comparable with that of the commercial enzymes. Therefore, the C2-2-1 β-galactosidase isoenzyme of Arthrobacter sp. C2-2 has the particular advantage that it could be used as a biotechnological tool in the production of lactose-reduced dairy products at refrigeration temperatures. [Copyright &y& Elsevier]

Abstract: The cluster of pbtTFYRABC genes is carried by plasmid pA81. Its elimination from Achromobacter xylosoxidans A8 resulted in increased sensitivity towards Pb2+ and Cd2+. Predicted pbtTRABC products share strong similarities with Pb2+ uptake transporter PbrT, transcriptional regulator PbrR, metal efflux P1-ATPases PbrA and CadA, undecaprenyl pyrophosphatase PbrB and its signal peptidase PbrC from Cupriavidus metallidurans CH34. Expression of pbtABC or pbtA in a metal-sensitive Escherichia coli GG48 rendered the strain Pb2+-, Cd2+- and Zn2+-tolerant and caused decreased accumulation of the metal ions. Accumulation of Pb2+, but not of Cd2+ or Zn2+, was promoted in E. coli expressing pbtT. Additional genes of the pbt cluster are pbtF and pbtY, which encode the cation diffusion facilitator (CDF)-like transporter and a putative fatty acid hydroxylase of unknown function, respectively. Expression of pbtF did not confer increased metal tolerance upon E. coli GG48, although the protein showed measurable Pb2+-efflux activity. Unlike the pbtT promoter, promoters of pbtABC, pbtF and pbtY contain features characteristic of promoters controlled by metal-responsive transcriptional regulators of the MerR family. Upregulation of pbtABC, pbtF and pbtY upon Pb2+, Cd2+ and Zn2+ exposure was confirmed in wild-type Achromobacter xylosoxidans A8. Gel shift assays proved binding of purified PbtR to the respective promoters. [Copyright &y& Elsevier]

Abstract Lepidopteran silk is a complex assembly of proteins produced by a pair of highly specialized labial glands called silk glands. Silk composition has been examined only in a handful of species. Here we report on the analysis of silk gland-specific transcriptomes from three developmental stages of the greater wax moth, Galleria mellonella, combined with proteomics, Edman microsequencing and northern blot analysis. In addition to the genes known earlier, we identified twenty seven candidate cDNAs predicted to encode secretory proteins, which may represent novel silk components. Eight were verified by proteomic analysis or microsequencing, and several others were confirmed by similarity with known silk genes and their expression patterns. Our results revealed that most candidates encode abundant secreted proteins produced by middle silk glands including ten sericins, two seroins, one or more mucins, and several sequences without apparent similarity to known proteins. We did not detect any novel PSG-specific protein, confirming that there are only three fibroin subunits. Our data not only show that the number of sericin genes in the greater wax moth is higher than in other species thus far examined, but also the total content of soluble proteins in silk is twice as high in G. mellonella than in B. mori or A. yamamai. Our data will serve as a foundation for future identification and evolutionary analysis of silk proteins in the Lepidoptera. Graphical abstract Image 1 Highlights • Soluble components of G. mellonella silk make up almost half of the total silk protein mass. • Seventeen novel highly or moderately expressed genes were characterized in detail. • Putative novel gene products include sericins, mucins and seroins. • This study improves our understanding of silk structure and evolution. [ABSTRACT FROM AUTHOR]

Mechanisms evolved in eukaryotes to handle heavy metals involve cytosolic, metal-binding metallothioneins (MTs). We have previously documented that the sequestration of silver (Ag) in the Ag-hyperaccumulating Amanita strobiliformis is dominated by 34-amino-acid (AA) AsMT1a, 1b, and 1c isoforms. Here we show that in addition to As MT1a , 1b , and 1c isogenes, the fungus has two other MT genes: As MT2 encoding a 34-AA AsMT2 similar to MTs known from other species, but unrelated to AsMT1s; As MT3 coding for a 62-AA AsMT3 that shares substantial identity with as-yet-uncharacterized conserved peptides predicted in agaricomycetes. Transcription of As MT1 s and As MT3 in the A. strobiliformis mycelium was specifically inducible by treatments with Ag or copper (Cu) and zinc (Zn) or cadmium (Cd), respectively; As MT2 showed a moderate upregulation in the presence of Cd. Expression of As MT s in the metal-sensitive Saccharomyces cerevisiae revealed that all As MT s confer increased Cd tolerance (As MT3 proved the most effective) and that, unlike As MT1 and As MT2 , As MT3 can protect the yeasts against Zn toxicity. The highest level of Cu tolerance was observed with yeasts expressing As MT1a . Our data indicate that A. strobiliformis can specifically employ different MT genes for functions in the cellular handling of Ag and Cu (As MT1 s) and Zn (As MT3 ). [ABSTRACT FROM AUTHOR]

Abstract: Malignant gliomas exhibit abnormal expression of proteolytic enzymes that may participate in the uncontrolled cell proliferation and aberrant interactions with the brain extracellular matrix. The multifunctional membrane bound serine aminopeptidase dipeptidyl peptidase (DPP)-IV has been linked to the development and progression of several malignancies, possibly both through the enzymatic and nonenzymatic mechanisms. In this report we demonstrate the expression of DPP-IV and homologous proteases fibroblast activation protein, DPP8 and DPP9 in primary cell cultures derived from high-grade gliomas, and show that the DPP-IV-like enzymatic activity is negatively associated with their in vitro growth. More importantly, the DPP-IV positive subpopulation isolated from the primary cell cultures using immunomagnetic separation exhibited slower proliferation. Forced expression of the wild as well as the enzymatically inactive mutant DPP-IV in glioma cell lines resulted in their reduced growth, migration and adhesion in vitro, as well as suppressed glioma growth in an orthotopic xenotransplantation mouse model. Microarray analysis of glioma cells with forced DPP-IV expression revealed differential expression of several candidate genes not linked to the tumor suppressive effects of DPP-IV in previous studies. Gene set enrichment analysis of the differentially expressed genes showed overrepresentation of gene ontology terms associated with cell proliferation, cell adhesion and migration. In conclusion, our data show that DPP-IV may interfere with several aspects of the malignant phenotype of glioma cells in great part independent of its enzymatic activity. [Copyright &y& Elsevier]