•We use iTRAQ-LC–MS/MS strategy for quantification of proteins in response to the stimuli of C18 alkane.•A global response is generated to support pseudomonads adapt to alkanes.•Alkane hydroxylase AlkB2 and a putative novel AlmA-like monooxygenase are contributed to alkane hydroxylation.•A full range of chemotaxic proteins are differentially expressed in alkane-grown cells.•We delineated an overall network of alkane bio-degradation.N-octadecane, the shortest solid-state alkane, was efficiently consumed by Pseudomonas aeruginosa SJTD-1. To reveal its mechanism, the iTRAQ-LC–MS/MS strategy was applied for quantification of proteins in response to alkane. As a result, 383 alkane-responsive proteins were identified and these proteins could be linked to multiple biochemical pathways. Above all, the level of alkane hydroxylase AlkB2 has been significantly higher in alkane condition. Also, the presence of a putative novel AlmA-like monooxygenase and its role on alkane hydroxylation were firstly proposed in Pseudomonas. In addition, other proteins for chemotaxic, β-oxidation, glyoxylate bypass, alkane uptake, cross membrane transport, enzymatic steps and the carbon flow may have important roles in the cellular response to alkane. Most of those differently expressed proteins were functionally mapped into pathways of alkane degradation or metabolism thereof. In this sense, findings in this study provide critical clues to reveal biodegradation of long chain n-alkanes and rationally be important for potent biocatalyst for bioremediation in future.Biological significanceWe use iTRAQ strategy firstly to compare the proteomes of Pseudomonas SJTD-1 degrading alkane. Changes in protein clearly provide a comprehensive overview on alkane hydroxylation of SJTD-1, including those proteins for chemotaxis, alkane uptake, cross membrane transport, enzymatic steps and the carbon flow. AlkB2 and a putative novel AlmA-like monooxygenase have been highlighted for their outstanding contribution to alkane use. We found that several chemotaxic proteins were altered in abundance in alkane-grown cells. These results may be helpful for understanding alkane use for Pseudomonas.

We recently provided the first report that RNase HIII can cleave a DNA-rN1-DNA/DNA substrate (rN1, one ribonucleotide) in vitro. In the present study, mutagenesis analyses and molecular dynamics (MD) simulations were performed on RNase HIII from Chlamydophila pneumoniae AR39 (CpRNase HIII). Our results elucidate the mechanism of ribonucleotide recognition employed by CpRNase HIII, indicating that the G95/K96/G97 motif of CpRNase HIII represents the main surface interacting with single ribonucleotides, in a manner similar to that of the GR(K)G motif of RNase HIIs. However, CpRNase HIII lacks the specific tyrosine required for RNase HII to recognize single ribonucleotides in double-stranded DNA (dsDNA). Interestingly, MD shows that Ser94 of CpRNase HIII forms a stable hydrogen bond with the deoxyribonucleotide at the (5')RNA–DNA(3') junction, moving this nucleotide away from the chimeric ribonucleotide. This movement appears to deform the nucleic acid backbone at the RNA–DNA junction and allows the ribonucleotide to interact with the GKG motif. Based on the inferences drawn from MD simulations, biochemical results indicated that Ser94 was necessary for catalytic activity on the DNA-rN1-DNA/DNA substrate; mutant S94V could bind this substrate but exhibited no cleavage. Mismatches opposite the single ribonucleotide misincorporated in dsDNA inhibited cleavage by CpRNase HIII to varying degrees but did not interfere with CpRNase/substrate binding. Further MD results implied that mismatches impair the interaction between Ser94 and the deoxyribonucleotide at the RNA–DNA junction. Consequently, recognition of the misincorporated ribonucleotide was disturbed. Our results may help elucidate the distinct substrate-recognition properties of different RNase Hs.Highlights► We construct a series of mutants of CpRNase HIII. ► We model the structure of CpRNase HIII complexed with DNA-rN1-DNA/DNA substrate. ► We elucidate the mechanism of ribonucleotide recognition employed by CpRNase HIII. ► Ser94 of CpRNase HIII plays an important role in recognizing single ribonucleotide.

Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation. SNPs are important markers that link sequence variations to phenotypic changes. Because of the importance of SNPs in the life and medical sciences, a great deal of effort has been devoted to developing accurate, rapid, and cost-effective technologies for SNP analysis. In this article, we describe a novel method for SNP genotyping based on differential fluorescence emission due to cleavage by Thermus thermophilus RNase HII (TthRNase HII) of DNA heteroduplexes containing an SNP site-specific chimeric DNA–rN1–DNA molecular beacon (cMB). We constructed a loop sequence for a cMB that contains a single SNP-specific ribonucleotide at the central site. When the cMB probe is hybridized to a target double-stranded DNA (dsDNA), a perfect match of the cMB/DNA duplex permits efficient cleavage with TthRNase HII, whereas a mismatch in the duplex due to an SNP greatly reduces efficiency. Cleavage efficiency is measured by the incremental difference of fluorescence emission of the beacon. We show that the genotypes of 10 individuals at 12 SNP sites across a series of human leukocyte antigen (HLA) can be determined correctly with respect to conventional DNA sequencing. This novel TthRNase HII-based method offers a platform for easy and accurate SNP analysis.

This article describes a new assay for isothermal enhancement of fluorescence intensity. The assay is based on the cleavage of duplexes formed by the chimeric DNA–rN1–DNA molecular beacon (cMB) and target DNA with Chlamydia pneumoniae RNase HII (CpRNase HII). The loop sequence of the cMB, which was designed according to the target sequence, contains a single ribonucleotide. The combination of CpRNase HII cleavage and cMB (RHMB) permitted a 90-fold increase in fluorescence intensity change compared with the hybridization reaction in the presence of the same amount of target DNA. These results indicate that the RHMB assay can enhance the fluorescence signal in real-time monitoring of the target DNA.

Chlamydophila pneumoniae AR39 is an obligate intracellular pathogen that causes human acute and chronic respiratory tract diseases. One protein from C. pneumoniae AR39 was assigned as 4-hydroxybenzoate decarboxylase (HBDC). Assays done with the purified oxygen-sensitive protein showed that the optimum pH and temperature were 7.5 and 30°C, respectively. The Km and Vmax obtained for 4-hydroxybenzoate were approximately 0.21 mM and 11.9 nM min−1 mg−1, respectively. During the period of 4-hydroxybenzoate decarboxylation, overall activity of the thermal-sensitive protein was 5.06 nM min−1 mg−1 protein. The 4-hydroxybenzoate decarboxylation was promoted by Mg2+, Fe2+, Mn2+, and Ca2+ but not by Cu2+ or Zn2+. The enzyme also slowly catalyzed the reverse reaction, which was phenol carboxylation.

Temperature-sensitive (TS) mutants of a gene are ones of which the activity or phenotype is very similar to that of wild type only at certain temperature and they provide extremely powerful tool for studying protein function in vivo. Here we report a novel strategy to generate TS phenotype of the interest gene in Escherichia coli based on a temperature-sensitive T7-expression system. A TS T7-RNA polymerase is generated by interrupting it with a TS intein from Saccharomyces cerevisiae vacuolar ATPase subunit (VMA), resulting that the gene flanked by T7-promoter and T7-terminator will be transcribed only at the permissive temperature (18 °C), not at the restrictive temperature (37 °C). The feasibility to create TS phenotype of this strategy was detected using lacZ as target. Reverse transcriptase polymerase chain reaction (PCR) indicated that at 18 °C, transcripts of T7-promoter controlled lacZ were at least 85 times more than those at 37 °C. Western blot analysis and enzymatic assay showed that large amounts of active His6-tagged LacZ produced at 18 °C but little at 37 °C. This strategy appears more promising than other TS creation methods because the target is pre-designed, no modification is introduced, and only simple DNA manipulation is required.

DNA polymerase I (DNApolI) catalyzes DNA synthesis during Okazaki fragment maturation, base excision repair, and nucleotide excision repair. Some bacterial DNApolIs are deficient in 3′–5′ exonuclease, which is required for removing an incorrectly incorporated 3′-terminal nucleotide during DNA elongation by DNA polymerase activity. The key amino acid residues in the exonuclease center of Chlamydophila pneumoniae DNApolI (CpDNApolI) are naturally mutated, resulting in the loss of 3′–5′ exonuclease. Hence, the manner by which CpDNApolI proofreads the incorrectly incorporated nucleotide during DNA synthesis warrants clarification. C. pneumoniae encodes three 3′–5′ exonuclease activities: one endonuclease IV and two homologs of the epsilon subunit of replicative DNA polymerase III. The three proteins were biochemically characterized using single- and double-stranded DNA substrate. Among them, C. pneumoniae endonuclease IV (CpendoIV) possesses 3′–5′ exonuclease activity that prefers to remove mismatched 3′-terminal nucleotides in the nick, gap, and 3′ recess of a double-stranded DNA (dsDNA). Finally, we reconstituted the proofreading reaction of the mismatched 3′-terminal nucleotide using the dsDNA with a nick or 3′ recess as substrate. Upon proofreading of the mismatched 3′-terminal nucleotide by CpendoIV, CpDNApolI can correctly reincorporate the matched nucleotide and the nick is further sealed by DNA ligase. Based on our biochemical results, we proposed that CpendoIV was responsible for proofreading the replication errors of CpDNApolI.Highlights► C. pneumoniae encodes only one endonuclease IV and two epsilon subunits of DNA polymerase III, which possess intrinsic 3′ exonuclease activity. ► The 3′–5′ exonuclease of C. pneumoniae endonuclease IV prefers to the mismatched 3′ nucleotides in the nick, gap, and 3′ recess of dsDNA. ► A proofreading system for mismatched 3′ nucleotide was constituted. ► C. pneumoniae endonuclease IV can efficiently proofread the mismatched 3′ nucleotides in the constituted proofreading system.