Variations in amino acid composition in bacterial single stranded DNA–binding proteins correlate with GC content

Background and purposeSSB proteins are essential for the maintenance of the genome in all domains of life. Most bacterial SSBs are active as homotetramers. Each monomer comprises N-terminal domain (OB-fold) which is responsible for ssDNA binding and a disordered C-terminal domain (Ct) with a conserved acidic tail responsible for protein interactions.The variations in these essential proteins prompted us to conduct in silico analyses of the aa composition and properties of two distinct SSB domains in relation to bacterial GC content.Materials and methodsSSB sequences were collected from genomes covering a wide range of GC content from 14 bacterial phyla. The maximum-likelihood (ML) trees were constructed for SSB sequences and corresponding 16S rRNA genes. The aa contents of OB folds and Ct domains were subsequently analysed. ResultsWe showed that SSB proteins followed predicted amino acid (aa) composition as a function of genomic GC content. However, two distinct domains of SSB exhibit significant differences to the expected aa composition. Variations in aa proportion were more prominent in Ct domains. Elevated accumulation of Gly (up to 60 %) and Pro (up to 24 %), significant drop in the proportion of basic Lys and reduction in hydrophobic Leu, Ile and Val were identified in Ct domains of SSBs from high GC genomes. Consequently, this influences the biochemical properties of Ct domains.ConclusionsBased on this comparative study of SSBs we conclude that genomic GC content and two distinct domains with different functional roles participate in shaping aa composition of SSB proteins.</p

Leitmotif: protein motif scanning 2.0

Motivation Motif-HMM (mHMM) scanning has been shown to possess unique advantages over standardly used sequence-profile search methods (e.g. HMMER, PSI-BLAST) since it is particularly well-suited to discriminate proteins with variations inside conserved motifs (e.g. family subtypes) or motifs lacking essential residues (false positives, e.g. pseudoenzymes). Results In order to make mHMM widely accessible to a broader scientific community, we developed Leitmotif, an mHMM web application with many parametrization options easily accessible through intuitive interface. Substantial improvement of performance (ROC scores) was obtained by using two novel parameters. To the best of our knowledge, Leitmotif is the only available mHMM applicatio

Streptomyces rimosus GDS(L) Lipase: Production, Heterologous Overexpression and Structure-Stability Relationship

Streptomyces rimosus lipase gene has been overexpressed in a heterologous host, S. lividans TK23. The maximal lipase activity was determined in the culture filtrates of the late stationary phase. Time course of lipase production was monitored by a modified plate assay. S. rimosus lipase gene has been located on the AseI B fragment approximately 2 Mb far from the left end of the S. rimosus linear chromosome. Out of eight examined streptomycetes, the presence of this rare type of bacterial lipase gene was detected in two belonging to the S. rimosus taxonomic cluster, and in one non-related species. Comparison of protein sequences of the Streptomyces lipolytic enzymes was performed. The result indicated the best structural stability of the putative S. coelicolor lipase-2

Preliminary Crystallographic Study of Streptomyces coelicolor Single-stranded DNA-binding Protein

Single-stranded DNA-binding proteins (SSBs) play a crucial role in DNA processing such as replication, repair and recombination in all organisms, from bacteria to human. Streptomyces coelicolor ssb gene was overexpressed in a heterologous host, Escherichia coli NM522. 15 mg of purified protein from 1 dm3 of culture was obtained in one-step procedure applying Ni2+ chelating chromatography. Among bacterial SSBs with the solved crystal structure, the S. coelicolor SSB displayed significant sequence similarity with those from Mycobacterium tuberculosis and Mycobacterium smegmatis, slow growing bacteria with a high GC content. Moreover, conserved amino acid region that forms additional ß strand in mycobacterial SSBs was also found in S. coelicolor SSB. The full-length protein readily crystallises in space group I222 or I212121 with unit-cell parameters a = 100.8, b = 102.1, c = 164.2 Å. The asymmetric unit is expected to contain four monomers with solvent content of 52–55 %

3'-terminated Overhangs Regulate DNA Double-Strand Break Processing in Escherichia coli

Double-strand breaks (DSBs) are lethal DNA lesions, which are repaired by homologous recombination in Escherichia coli To study DSB processing in vivo, we induced DSBs into the E. coli chromosome by gamma irradiation and measured chromosomal degradation. We show that the DNA degradation is regulated by RecA protein concentration and its rate of association with ssDNA. RecA decreased DNA degradation in wild- type, recB and recD strains, indicating that it is a general phenomenon in E. coli On the other hand, DNA degradation was greatly reduced and unaffected by RecA in the recB1080 mutant (which produces long overhangs) and in a strain devoid of four exonucleases that degrade a 3' tail (ssExos). 3'-5' ssExos deficiency is epistatic to RecA deficiency concerning DNA degradation, suggesting that bound RecA is shielding 3' tail from degradation by 3'-5' ssExos. Since 3'-tail preservation is common to all these situations, we infer that RecA polymerization constitutes a subset of mechanisms for preserving the integrity of 3' tails emanating from DSBs, along with 3' tail's massive length, or prevention of their degradation by inactivation of 3'-5' ssExos. Thus, we conclude that 3' overhangs are crucial in controlling the extent of DSB processing in E. coli This study suggests a regulatory mechanism for DSB processing in E. coli, wherein 3' tails impose a negative feedback loop on DSB processing reactions, specifically on helicase reloading onto dsDNA ends

Clustering of protein domains for functional and evolutionary studies

Background: The number of protein family members defined by DNA sequencing is usually much larger than those characterised experimentally. This paper describes a method to divide protein families into subtypes purely on sequence criteria. Comparison with experimental data allows an independent test of the quality of the clustering. Results: An evolutionary split statistic is calculated for each column in a protein multiple sequence alignment; the statistic has a larger value when a column is better described by an evolutionary model that assumes clustering around two or more amino acids rather than a single amino acid. The user selects columns (typically the top ranked columns) to construct a motif. The motif is used to divide the family into subtypes using a stochastic optimization procedure related to the deterministic annealing EM algorithm (DAEM), which yields a specificity score showing how well each family member is assigned to a subtype. The clustering obtained is not strongly dependent on the number of amino acids chosen for the motif. The robustness of this method was demonstrated using six well characterized protein families: nucleotidyl cyclase, protein kinase, dehydrogenase, two polyketide synthase domains and small heat shock proteins. Phylogenetic trees did not allow accurate clustering for three of the six families. Conclusion: The method clustered the families into functional subtypes with an accuracy of 90 to 100%. False assignments usually had a low specificity score