Complete genome and proteome of Acholeplasma laidlawii.

We present the complete genome sequence and proteogenomic map for Acholeplasma laidlawii PG-8A (class Mollicutes, order Acholeplasmatales, family Acholeplasmataceae). The genome of A. laidlawii is represented by a single 1,496,992-bp circular chromosome with an average G+C content of 31 mol%. This is the longest genome among the Mollicutes with a known nucleotide sequence. It contains genes of polymerase type I, SOS response, and signal transduction systems, as well as RNA regulatory elements, riboswitches, and T boxes. This demonstrates a significant capability for the regulation of gene expression and mutagenic response to stress. Acholeplasma laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code, in which UGA is a stop codon. Within the Mollicutes group, only the sterol-nonrequiring Acholeplasma has the capacity to synthesize saturated fatty acids de novo. Proteomic data were used in the primary annotation of the genome, validating expression of many predicted proteins. We also detected posttranslational modifications of A. laidlawii proteins: phosphorylation and acylation. Seventy-four candidate phosphorylated proteins were found: 16 candidates are proteins unique to A. laidlawii, and 11 of them are surface-anchored or integral membrane proteins, which implies the presence of active signaling pathways. Among 20 acylated proteins, 14 contained palmitic chains, and six contained stearic chains. No residue of linoleic or oleic acid was observed. Acylated proteins were components of mainly sugar and inorganic ion transport systems and were surface-anchored proteins with unknown functions.

Proteins and peptides of the salivary gland secretion of medicinal leeches Hirudo verbana, H. medicinalis, and H. orientalis.

The protein and peptide composition of medicinal leech salivary gland secretion (SGS) was analyzed in preparations obtained in July from three species--Hirudo verbana, H. medicinalis, and H. orientalis. Two-dimensional electrophoresis (molecular mass 10-150 kD and pI 3-10) revealed no distinctions in the distribution of over 100 silver-stained proteins. Differences were noted only in intensity of 10 protein spots at 30-90 kD and pI 4.7-7.5. Mass spectrometric profiling of SGS of the three leech species using the Zip-Tip/golden chip scheme and cation-exchanging chips CM-10 revealed over 50 components in SGS of each of the three leech species. It was noted that 30-40% of the individual masses of the SGS of each leech species fall within the masses present in SGS of at least one of the two other species. This rather small part of the total mass may be indicative of a high polymorphism of amino acid sequences or a high frequency of posttranslational modifications of the SGS proteins and peptides. Calculation of Jacquard's coefficient showed that H. medicinalis and H. orientalis are closest to each other in SGS composition, which is consistent with data in the literature on the phylogenetic relationship between these two species of medicinal leech. Comparison of detected molecular masses with those of six known biologically active compounds produced by medicinal leeches revealed their uneven distribution in SGS of each of the three medicinal leech species. This opens prospects for using certain species of medicinal leech for targeted therapy of various pathologies.

Localization of C. trachomatis Inc proteins in expression of their genes in HeLa cell culture.

Co-localization of Chlamydia trachomatis incorporation membrane proteins with cell organelles was studied in HeLa cell culture after transfection by expressing vectors carrying incA, incB, incC, incD, incE, incF, incG genes, respectively, fused with the marker green fluorescent protein (EGFP) gene. The prokaryotic proteins were co-located with compartments of the secretory pathway of the eukaryotic cell in the course of biogenesis.

Proteomic analysis methods for characterization of proteins from the salivary gland secretions of the medicinal leech during different seasons.

Salivary gland secretion (SGS) of the medicinal leech Hirudo medicinalis in summer and winter was studied by proteomic analysis methods, and season-associated difference was found in the distribution of fractionated proteins with the same pattern of their positions. Differences were detected for proteins with molecular weights from 15 to 250 kD fractionated by two-dimensional SDS-PAGE and for 2-10- and 10-60-kD proteins analyzed by SELDI-MS. Thirty-two and 20 proteins were detected by MALDI-TOF-MS in the high-molecular-weight fraction of the summer and winter SGS, respectively, isolated from the corresponding two-dimensional electrophoregrams, and this was less than 20% of the total SGS protein. The N-terminal amino acid sequences were determined for 12 proteins. The peptide maps and N-terminal amino acid sequences of the proteins studied were identified, and no known proteins were revealed. These findings suggest a high content of newly revealed proteins in SGS of medicinal leech, and this correlates with multiple positive clinical effects of hirudotherapy realized through SGS, but the mechanisms of these effects remain unclear.

Expression of Chlamydia trachomatis inclusion membrane protein genes IncB and IncC in Escherichia coli.

In this study, we have cloned the Chlamydia trachomatis genes incB and incC into the expression plasmid vectors from pET series for the subsequent isolation of recombinant proteins. As a result, we have obtained the first full-length recombinant C. trachomatis proteins IncB and IncC, which can be used for following antibody production and for study of their protein-protein interaction.

Effect of induced expression of an antimicrobial peptide melittin on Chlamydia trachomatis and Mycoplasma hominis infections in vivo.

A plasmid construct was designed in which the gene of antimicrobial peptide melittin is controlled by the tetracycline-responsive promoter of human cytomegalovirus, aided by a constitutively expressed trans-activator protein gene. Its vaginal administration and induction of melittin gene transcription with doxycycline markedly suppressed subsequent genital tract infection of mice by Mycoplasma hominis and Chlamydia trachomatis. At least half of the melittin-protected animals proved free of either pathogen within 3-4 weeks. Recombinant plasmids expressing genes of antimicrobial peptides hold much promise as agents for prevention and control of urogenital latent infections.