Impact of Protein Domains on PE_PGRS30 Polar Localization in Mycobacteria

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.     

A novel protease from Entamoeba histolytica homologous to members of the family S28 of serine proteases

Serine proteases are one of the biologically most important and widely distributed enzyme families. A protease capable of degrading the substrate Suc-AAF-AMC was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified by ion-exchange chromatography and electroelution, and appeared on 2D-PAGE as a spot of 60 kDa and pI of 4.65. Data obtained from zymogram suggest the active protease is present either as homodimer (130 kDa) or homotetramer (250 kDa). The optimal temperature of the enzyme was 37 degrees C, and it exhibited activity over a broad pH range. The protease was strongly inhibited by TPCK and chelating agents. The enzymatic activity was restored upon addition of calcium. BLAST analysis with the sequence of internal peptides of the protein revealed two open reading frames within the genome of E. histolytica, homologous to members of the family S28, clan SC of serine proteases.     

Quantitative proteomic analysis of drug-induced changes in mycobacteria

A new approach for qualitative and quantitative proteomic analysis using capillary liquid chromatography and mass spectrometry to study the protein expression response in mycobacteria following isoniazid treatment is discussed. In keeping with known effects on the fatty acid synthase II pathway, proteins encoded by the kas operon (AcpM, KasA, KasB, Accd6) were significantly overexpressed, as were those involved in iron metabolism and cell division suggesting a complex interplay of metabolic events leading to cell death.     

The isolation of alpha-1-protease inhibitor by a unique procedure designed for industrial application

Individuals who are congenitally deficient in the human plasma protein α₁-protease inhibitor (α₁PI, which is also called α₁-antitrypsin) usually develop chronic obstructive lung disease as a consequence of improperly regulated granulocyte elastase. In this report, a unique, facile one- or two-step method is presented for the large-scale isolation of α₁PI for potential therapeutic use. The method takes advantage of the unusual disulfide bond in α₁PI, which consists of a single cysteine residue in the polypeptide chain bound to a free pendant cysteine. In contrast to other circulating plasma proteins, the disulfide bridge in α₁PI does not add to its structural stability. Therefore, if an α₁PI-containing solution of plasma proteins is precipitated out in the presence of reductant, much more extensive separation of contaminating proteins will be achieved than in the absence of such reductant. We have used Cohn Fraction IV-1, a relatively unused side product in albumin and gamma globulin production, as our starting material. After activation of the α₁PI in basic media, partial purification is achieved with successive additions of Aerosil (a fumed silica), dithiothreitol, and ammonium sulfate. From 90 to 95% of the contaminating proteins are precipitated by this single procedure, resulting in a product that is ～70% pure. DEAE-cellulose chromatography can be used as an additional purification step, and this results in a product that is nearly homogenous. Overall yield is ～45%. The method is simple, inexpensive, and reproducible and is directly applicable to large-scale industrial processing.     

Influence of urea and farmyard manure compost as N source on symbiotic association between <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> (MTTC 10096)<Emphasis Type="Italic">—Neptunia prostrata</Emphasis> (Lam.) Baill

Growth, biomass production, nitrogen fixation and nodulation in Neptunia prostrata (Lam.) Baill. is affected differently by various N sources and concentrations but the subject needs to be discussed in more depth. Effects of urea and farmyard manure compost (FYMC) as N source on the symbiotic association between Rhizobium leguminosarum MTTC10096—Neptunia prostrata (Lam.) Baill., biomass production, nitrogen accumulation, etc., at three different concentrations in loamy soil culture were investigated. The total N in both urea and FYMC were of equal proportion, but FYMC was more beneficial in overall performance of Neptunia prostrata plant. Nodule number and biomass were greatly depressed but plant length and weight showed slight increase by the higher N concentration in all the samplings. The study revealed that higher concentration of urea has more negative effect on nodule biomass and number than FYMC as compared to control. The plants also showed better N percent accumulation on the application of FYMC than urea. The overall performance of the plant was more pronounced on FYMC application as N source. It might be due to the presence of other vital nutrients in FYMC other than N, essential for both the host plant and microsymbiont. This aspect, however, awaits further experimental proof.     

Epigenetic natural variation in Arabidopsis thaliana

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F₂ families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.