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.
Pseudomonas aeruginosa phage EL is a dsDNA phage related to the giant ϕKZ-like Myoviridae. The EL genome sequence comprises 211,215
bp and has 201 predicted open reading frames (ORFs). The EL genome ...does not share DNA sequence homology with other viruses and micro-organisms sequenced to date. However, one-third of the predicted EL gene products (gps) shares similarity (Blast alignments of 17–55% amino acid identity) with ϕKZ proteins. Comparative EL and ϕKZ genomics reveals that these giant phages are an example of substantially diverged genetic mosaics. Based on the position of similar EL and ϕKZ predicted gene products, five genome regions can be delineated in EL, four of which are relatively conserved between EL and ϕKZ. Region IV, a 17.7
kb genome region with 28 predicted ORFs, is unique to EL. Fourteen EL ORFs have been assigned a putative function based on protein similarity. Assigned proteins are involved in DNA replication and nucleotide metabolism (NAD
+-dependent DNA ligase, ribonuclease HI, helicase, thymidylate kinase), host lysis and particle structure. EL-gp146 is the first chaperonin GroEL sequence identified in a viral genome. Besides a putative transposase, EL harbours predicted mobile endonucleases related to H–N–H and LAGLIDADG homing endonucleases associated with group I intron and intein intervening sequences.
DNA-binding transcription factors (TFs) are essential components of transcriptional regulatory networks in bacteria. LacI-family TFs (LacI-TFs) are broadly distributed among certain lineages of ...bacteria. The majority of characterized LacI-TFs sense sugar effectors and regulate carbohydrate utilization genes. The comparative genomics approaches enable in silico identification of TF-binding sites and regulon reconstruction. To study the function and evolution of LacI-TFs, we performed genomics-based reconstruction and comparative analysis of their regulons. For over 1300 LacI-TFs from over 270 bacterial genomes, we predicted their cognate DNA-binding motifs and identified target genes. Using the genome context and metabolic subsystem analyses of reconstructed regulons, we tentatively assigned functional roles and predicted candidate effectors for 78 and 67% of the analyzed LacI-TFs, respectively. Nearly 90% of the studied LacI-TFs are local regulators of sugar utilization pathways, whereas the remaining 125 global regulators control large and diverse sets of metabolic genes. The global LacI-TFs include the previously known regulators CcpA in Firmicutes, FruR in Enterobacteria, and PurR in Gammaproteobacteria, as well as the three novel regulators-GluR, GapR, and PckR-that are predicted to control the central carbohydrate metabolism in three lineages of Alphaproteobacteria. Phylogenetic analysis of regulators combined with the reconstructed regulons provides a model of evolutionary diversification of the LacI protein family. The obtained genomic collection of in silico reconstructed LacI-TF regulons in bacteria is available in the RegPrecise database (http://regprecise.lbl.gov). It provides a framework for future structural and functional classification of the LacI protein family and identification of molecular determinants of the DNA and ligand specificity. The inferred regulons can be also used for functional gene annotation and reconstruction of sugar catabolic networks in diverse bacterial lineages.
Bacteriophages of the family Myoviridae represent one of the most widespread domains of the biosphere substantially affecting the ecological balance of microorganisms. Interestingly, sequence ...analysis of genomic DNAs of large bacteriophages revealed many genes coding for proteins with unknown functions. A new approach is proposed to improve the functional identification of genes. This approach is based on comparing the genome sequence for phylogenetically and morphologically related phages showing no considerable homology at the level of genomic DNA. It is assumed that gene functions essential for the development of phages of a given family are conserved and that the corresponding genes code for similar orthologous proteins even when lacking sequence homology. The genome was sequenced and compared for two Pseudomonas aeruginosa giant bacteriophages, phi KZ and EL, which belong to a group of phi KZ-related phages. A substantial difference in genome organization was observed, suggesting specific features of phage evolution. In addition, the problem of the minimal genome of the superfamily is discussed on the basis of the difference in size and structure between the phi KZ and EL genomes.
Fibritin is a segmented coiled‐coil homotrimer of the 486‐residue product of phage T4 gene wac. This protein attaches to a phage particle by the N‐terminal region and forms fibrous whiskers of 530 Å, ...which perform a chaperone function during virus assembly. The short C‐terminal region has a β‐annulus‐like structure. We engineered a set of fibritin deletion mutants sequentially truncated from the N‐termini, and the mutants were studied by differential scanning calorimetry (DSC) and CD measurements. The analysis of␣DSC curves indicates that full‐length fibritin exhibits three␣thermal‐heat‐absorption peaks centred at 321 K (ΔH=1390 kJ·mol trimer−1), at 336 K (ΔH=7600 kJ·mol trimer−1), and at 345 K (ΔH=515 kJ·mol trimer−1). These transitions were assigned to the N‐terminal, segmented coiled‐coil, and C‐terminal functional domains, respectively. The coiled‐coil region, containing 13 segments, melts co‐operatively as a single domain with a mean enthalpy ΔHres=21 kJ·mol residue−1. The ratio of ΔHVH/ΔHcal for the coiled‐coil part of the 120‐, 182‐, 258‐ and 281‐residue per monomer mutants, truncated from the N‐termini, and for full‐length fibritin are 0.91, 0.88, 0.42, 0.39, and 0.13, respectively. This gives an indication of the decrease of the ‘all‐or‐none’ character of the transition with increasing protein size. The deletion of the 12‐residue‐long loop in the 120‐residue fibritin increases the thermal stability of the coiled‐coil region. According to CD data, full‐length fibritin and all the mutants truncated from the N‐termini refold properly after heat denaturation. In contrast, fibritin XN, which is deleted for the C‐terminal domain, forms aggregates inside the cell. The XN protein can be partially refolded by dilution from urea and does not refold after heat denaturation. These results confirm that the C‐terminal domain is essential for correct fibritin assembly both in vivo and in vitro and acts as a foldon.
Gene product 12 of bacteriophage T4, adhesin, serves to adhere the virus to host cells. Adhesin is a fibrous homotrimer, and a novel tertiary structure element, a beta-helix, is supposed to be a ...major structural feature of this protein. We have constructed two truncated gp12 mutants, 12N1 and 12N2, containing 221 and 135 N-terminal residues, respectively. When expressed in E. coli cells, these gp12 fragments formed labile beta-structural trimers. Another hybrid protein, 12FN, containing 179 N-terminal amino acid residues of gp12 fused to the C-terminal domain (31 amino acids) of T4 fibritin, was shown to have a trimeric proteolytically resistant alpha-helical structure. This structure is probably similar to that of fibritin, which has a triple alpha-helical coiled-coil structure. Hence, we have demonstrated the possibility of global transformation of fibrous protein structure using fusion with a C-terminal domain that initiates trimerization.
Bacteriophages of the family Myoviridae represent one of the most widespread domains of the biosphere substantially affecting the ecological balance of microorganisms. Interestingly, sequence ...analysis of genomic DNAs of large bacteriophages revealed many genes coding for proteins with unknown functions. A new approach is proposed to improve the functional identification of genes. This approach is based on comparing the genome sequence for phylogenetically and morphologically related phages showing no considerable homology at the level of genomic DNA. It is assumed that gene functions essential for the development of phages of a given family are conserved and that the corresponding genes code for similar orthologous proteins even when lacking sequence homology. The genome was sequenced and compared for two Pseudomonas aeruginosa giant bacteriophages, phiKZ and EL, which belong to a group of (phiKZ-related phages. A substantial difference in genome organization was observed, suggesting specific features of phage evolution. In addition, the problem of the minimal genome of the superfamily is discussed on the basis of the difference in size and structure between the phiKZ and EL genomes.
Gene product 12 of bacteriophage T4, adhesin, serves to adhere the virus to host cells. Adhesin is a fibrous homotrimer, and a novel tertiary structure element, a β-helix, is supposed to be a major ...structural feature of this protein. We have constructed two truncated gp12 mutants, 12N1 and 12N2, containing 221 and 135 N-terminal residues, respectively. When expressed in E. coli cells, these gp12 fragments formed labile β-structural trimers. Another hybrid protein, 12FN, containing 179 N-terminal amino acid residues of gp12 fused to the C-terminal domain (31 amino acids) of T4 fibritin, was shown to have a trimeric proteolytically resistant a-helical structure. This structure is probably similar to that of fibritin, which has a triple α-helical coiled-coil structure. Hence, we have demonstrated the possibility of global transformation of fibrous protein structure using fusion with a C-terminal domain that initiates trimerization.PUBLICATION ABSTRACT
Gene product 12 of bacteriophage T4, adhesin, serves to adhere the virus to host cells. Adhesin is a fibrous homotrimer, and a novel tertiary structure element, a b-helix, is supposed to be a major ...structural feature of this protein. We have constructed two truncated gp12 mutants, 12N1 and 12N2, containing 221 and 135 N-terminal residues, respectively. When expressed in E. coli cells, these gp12 fragments formed labile b-structural trimers. Another hybrid protein, 12FN, containing 179 N-terminal amino acid residues of gp12 fused to the C-terminal domain (31 amino acids) of T4 fibritin, was shown to have a trimeric proteolytically resistant a-helical structure. This structure is probably similar to that of fibritin, which has a triple a-helical coiled-coil structure. Hence, we have demonstrated the possibility of global transformation of fibrous protein structure using fusion with a C-terminal domain that initiates trimerization.
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