Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase.

The neutral metalloprotease elastase is one of the major proteins secreted into the culture medium by many Pseudomonas aeruginosa strains. Encoded by the lasB gene, the 33-kDa elastase is initially synthesized as a 53-kDa preproenzyme which is processed to the mature form via a 51-kDa proelastase intermediate. To facilitate studies on proteolytic processing of elastase precursors and on secretion, we developed systems for overexpression of lasB in Escherichia coli under the control of the inducible T7 and tac promoters. Although the 51-kDa proelastase form was detectable in E. coli under inducible conditions, most of the elastase produced under these conditions was found in an enzymatically active 33-kDa form. The amino-terminal sequence of the first 15 amino acid residues of this 33-kDa elastase species was identical to that of the mature P. aeruginosa enzyme, suggesting that processing was autocatalytic. To test this possibility, the codon in lasB encoding His-223, a presumed active-site residue, was changed to encode Asp-223 (lasB1) and Tyr-223 (lasB2). The effects of these mutations on enzyme activity and processing were examined. No proteolytic or elastolytic activities were detected in extracts of E. coli cells containing the lasB mutant alleles. Overexpression of the mutated lasB genes in E. coli resulted in the accumulation of the corresponding 51-kDa proelastase species. These were processed in vitro to the respective 33-kDa forms by incubation with exogenous purified elastase, without an increase in proteolytic activity. Molecular modeling studies suggest that the mutations have little or no effect on the conformation of the mutant elastases. In addition, wild-type elastase and the mutant proelastases were localized to the periplasm of E. coli. The present results confirm that His-223 is essential for elastase activity and provide evidence for autoproteolytic processing of proelastase.     

Assemblage structure,vertical distributions and stable-isotope compositions of anguilliform leptocephali in the Gulf of Mexico

In August 2007, October 2008 and September–October 2010, 241 Tucker trawl and plankton net tows were conducted at the surface to depths of 1377 m at six locations in the northern and eastern Gulf of Mexico (GOM) to document leptocephalus diversity and determine how assemblage structure, larval size, abundance and isotopic signatures differ across the region and with depth. Overall, 2696 leptocephali representing 59 distinct taxa from 10 families were collected. Five families accounted for 96% of the total catch with Congridae and Ophichthidae being the most abundant. The top four most abundant species composed 59% of the total catch and included: Ariosoma balearicum, Paraconger caudilimbatus, Rhynchoconger flavus and Ophichthus gomesii. Four anguilliform species not previously documented in the GOM as adults or leptocephali were collected in this study, including Monopenchelys acuta, Quassiremus ascensionis, Saurenchelys stylura and one leptocephalus only known from its larval stage, Leptocephalus proboscideus. Leptocephalus catches were significantly greater at night than during the day. Catches at night were concentrated in the upper 200 m of the water column and significantly declined with increasing depth. Leptocephali abundances and assemblages were significantly different between sites on the upper continental slope (c. 500 m depth) and sites on the middle to lower continental slope (c. 1500–2300 m). Sites on the lower continental slope had a mixture of deep-sea demersal, bathypelagic and coastal species, whereas upper-slope sites contained several numerically dominant species (e.g., A. balearicum, P. caudilimbatus) that probably spawn over the continental shelf and upper slope of the GOM. Standard lengths of the four dominant species differed between sites and years, indicating heterochronic reproduction and potential larval source pools within and outside of the GOM. Stable-isotope analyses (δ¹³C and δ¹⁵N) conducted on 185 specimens from six families revealed that leptocephali had a wide range of isotopic values at the family and size-class levels. Species in the families Muraenidae, Congridae and Ophichthidae had similar δ¹⁵N values compared with the broad range of δ¹⁵N values seen in the deep-sea families Nemichthyidae, Nettastomatidae and Synaphobranchidae. Stable-isotope values were variably related to length, with δ¹⁵N values being positively size correlated in ophichthids and δ¹³C values being negatively size correlated in A. balearicum and P. caudilimbatus. Results suggest that leptocephali feed in various water depths and masses, and on different components of POM, which could lead to niche partitioning. Ecological aspects of these important members of the plankton community provide insight into larval connectivity in the GOM as well as the early life history of Anguilliformes.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Virulence factor regulation and regulatory networks in Streptococcus pyogenes and their impact on pathogen-host interactions

Streptococcus pyogenes (group A streptococcus, GAS) is a very important human pathogen with remarkable adaptation capabilities. Survival within the harsh host surroundings requires sensing potential on the bacterial side, which leads in particular to coordinately regulated virulence factor expression. GAS 'stand-alone' response regulators (RRs) and two-component signal transduction systems (TCSs) link the signals from the host environment with adaptive responses of the bacterial cell. Numerous putative regulatory systems emerged from GAS genome sequences. Only three RRs [Mga, RofA-like protein (RALP) and Rgg/RopB] and three TCSs (CsrRS/CovRS, FasBCAX and Ihk/Irr) have been studied in some detail with respect to their growth-phase-dependent activity and their influence on GAS-host cell interaction. In particular, the Mga-, RALP- and Rgg/RopB-regulated pathways display interconnected activities that appear to influence GAS colonization, persistence and spreading mechanisms, in a growth-phase-related fashion. Here, we have summarized our current knowledge about these RRs and TCSs to highlight the questions that should be addressed in future research on GAS pathogenicity.     

Two DNA-binding domains of Mga are required for virulence gene activation in the group A streptococcus

Mga is a DNA-binding protein that activates expression of several important virulence genes in the group A streptococcus (GAS), including those encoding M protein (emm), C5a peptidase (scpA) and Mga (mga). To determine the functionality of four potential helix-turn-helix DNA-binding motifs (HTH1-HTH4) identified within the amino-terminus of Mga, alanine substitutions were introduced within each domain in a MBP-Mga fusion allele and purified proteins were assayed for binding to Mga-specific promoter fragments (Pmga, PscpA and Pemm) in vitro. Although HTH-1 and HTH-2 mutations showed wild type DNA-binding activity, an altered HTH-3 domain resulted in reduced binding to the three promoters and an HTH-4 mutant was devoid of detectable binding activity. Plasmid-encoded expression of the HTH-3 and HTH-4 alleles from a constitutive promoter (Pspac) in the mga-deleted GAS strain JRS519 demonstrated that Mga-regulated emm expression correlated directly to the DNA-binding activity observed for each mutant protein in vitro. Single-copy expression of HTH-3 and HTH-4 from their native Pmga resulted in a dramatic reduction in autoregulated mga expression in both mutant strains. Thus, Mga appears to contain two DNA-binding domains (HTH-3 and HTH-4) that are required for direct activation of the Mga virulence regulon in vivo.     

Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.     

Apoptosis,cytokine and chemokine induction by non-structural 1 (NS1) proteins encoded by different influenza subtypes

Background

Influenza pandemic remains a serious threat to human health. Viruses of avian origin, H5N1, H7N7 and H9N2, have repeatedly crossed the species barrier to infect humans. Recently, a novel strain originated from swine has evolved to a pandemic. This study aims at improving our understanding on the pathogenic mechanism of influenza viruses, in particular the role of non-structural (NS1) protein in inducing pro-inflammatory and apoptotic responses.

Methods

Human lung epithelial cells (NCI-H292) was used as an in-vitro model to study cytokine/chemokine production and apoptosis induced by transfection of NS1 mRNA encoded by seven infleunza subtypes (seasonal and pandemic H1, H2, H3, H5, H7, and H9), respectively.

Results

The results showed that CXCL-10/IP10 was most prominently induced (> 1000 folds) and IL-6 was slightly induced (< 10 folds) by all subtypes. A subtype-dependent pattern was observed for CCL-2/MCP-1, CCL3/MIP-1α, CCL-5/RANTES and CXCL-9/MIG; where induction by H5N1 was much higher than all other subtypes examined. All subtypes induced a similar temporal profile of apoptosis following transfection. The level of apoptosis induced by H5N1 was remarkably higher than all others. The cytokine/chemokine and apoptosis inducing ability of the 2009 pandemic H1N1 was similar to previous seasonal strains.

Conclusions

In conclusion, the NS1 protein encoded by H5N1 carries a remarkably different property as compared to other avian and human subtypes, and is one of the keys to its high pathogenicity. NCI-H292 cells system proves to be a good in-vitro model to delineate the property of NS1 proteins.

     

Microbial oxidation of naphthalene to cis-1,2-naphthalene dihydrodiol using naphthalene dioxygenase in biphasic media

The selective oxidation of aryl substrates to chiral cis-1,2-dihydrodiols is an industrially important reaction for the production of intermediates that can be used to produce fine chemicals, pharmaceuticals, and many other bioactive natural products. More specifically, the oxidation of naphthalene to produce optically pure (+)-cis-(1R,2S)-1,2-napthalene dihydrodiol (NDHD) to be used as a chiral synthon for specialty chemicals has gained much interest. Escherichia coli JM109(DE3) pDTG141 expresses naphthalene dioxygenase which catalyzes this reaction. Poor substrate solubility and substrate toxicity are barriers to using the power of these enzymes in large-scale aqueous whole cell systems. A biphasic reaction system was chosen to overcome these barriers. The optimal biphasic conditions for E. coli JM109(DE3) pDTG141 were determined to be 20% dodecane as the organic solvent containing 40 g/L naphthalene. The productivity of the biotransformation using resting cells was 1.75 g-diol/g-cdw/h for the first 6 h with 20% organic phase, which was increased from 0.59 g-diol/g-cdw/h for growing cells with 40% organic phase. The biocatalytic activity was retained for at least 12 h. The biocatalyst could be recycled for at least four runs in both suspended and immobilized form. The stability of the 12 h recycle was improved by immobilization in calcium alginate beads. The process has been improved both environmentally and economically by reducing the amount of solvent used and by recycling the biocatalyst.     

Observations on the role of simuliids and culicids in the transmission of avian and anuran trypanosomes

Although Trypanosoma rotatorium undergoes transformation and prolific multiplication in the mid and hindgut of Culex territans, several attempts to experimentally infect laboratory-reared, Rana pipiens by different routes were unsuccessful. Trypanosoma avium undergoes a similar pattern of development in the gut of Simulium rugglesi. Experimental infections of T. avium in ducklings indicates that S. rugglesi serves as an intermediate host and probably as an active and passive vector of the parasite in nature.