Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both Bacillus subtilis and Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Phospholipid biosynthesis commences with the acylation of glycerol-3-phosphate (G3P) to form 1-acyl-G3P. This step is catalyzed by the PlsB protein in <it>Escherichia coli</it>. The gene encoding this protein has not been identified, however, in the majority of bacterial genome sequences, including that of <it>Bacillus subtilis</it>. Recently, a new two-step pathway catalyzed by PlsX and PlsY proteins for the initiation of phospholipid formation in <it>Streptococcus pneumoniae </it>has been reported.</p> <p>Results</p> <p>In <it>B. subtilis</it>, 271 genes have been reported to be indispensable, when inactivated singly, for growth in LB medium. Among these, 11 genes encode proteins with unknown functions. As part of a genetic study to identify the functions of these genes, we show here that the <it>B. subtilis </it>ortholog of <it>S. pneumoniae </it>PlsY, YneS, is required for G3P acyltransferase activity, together with PlsX. The <it>B. subtilis </it>genome lacks <it>plsB</it>, and we show in vivo that the PlsX/Y pathway is indeed essential for the growth of bacteria lacking <it>plsB</it>. Interestingly, in addition to <it>plsB</it>, <it>E. coli </it>possesses <it>plsX </it>and the <it>plsY </it>ortholog, <it>ygiH</it>. We therefore explored the functional relationship between PlsB, PlsX and YgiH in <it>E. coli</it>, and found that <it>plsB </it>is essential for <it>E. coli </it>growth, indicating that PlsB plays an important role in 1-acyl-G3P synthesis in <it>E. coli</it>. We also found, however, that the simultaneous inactivation of <it>plsX </it>and <it>ygiH </it>was impossible, revealing important roles for PlsX and YgiH in <it>E. coli </it>growth.</p> <p>Conclusion</p> <p>Both <it>plsX </it>and <it>yneS </it>are essential for 1-acyl-G3P synthesis in <it>B. subtilis</it>, in agreement with recent reports on their biochemical functions. In <it>E. coli</it>, PlsB plays a principal role in 1-acyl-G3P synthesis and is also essential for bacterial growth. PlsX and YgiH also, however, play important roles in <it>E. coli </it>growth, possibly by regulating the intracellular concentration of acyl-ACP. These proteins are therefore important targets for development of new antibacterial agents.</p

AMDORAP: Non-targeted metabolic profiling based on high-resolution LC-MS

<p>Abstract</p> <p>Background</p> <p>Liquid chromatography-mass spectrometry (LC-MS) utilizing the high-resolution power of an orbitrap is an important analytical technique for both metabolomics and proteomics. Most important feature of the orbitrap is excellent mass accuracy. Thus, it is necessary to convert raw data to accurate and reliable <it>m/z </it>values for metabolic fingerprinting by high-resolution LC-MS.</p> <p>Results</p> <p>In the present study, we developed a novel, easy-to-use and straightforward <it>m/z </it>detection method, AMDORAP. For assessing the performance, we used real biological samples, <it>Bacillus subtilis </it>strains 168 and MGB874, in the positive mode by LC-orbitrap. For 14 identified compounds by measuring the authentic compounds, we compared obtained <it>m/z </it>values with other LC-MS processing tools. The errors by AMDORAP were distributed within ±3 ppm and showed the best performance in <it>m/z </it>value accuracy.</p> <p>Conclusions</p> <p>Our method can detect <it>m/z </it>values of biological samples much more accurately than other LC-MS analysis tools. AMDORAP allows us to address the relationships between biological effects and cellular metabolites based on accurate <it>m/z </it>values. Obtaining the accurate <it>m/z </it>values from raw data should be indispensable as a starting point for comparative LC-orbitrap analysis. AMDORAP is freely available under an open-source license at <url>http://amdorap.sourceforge.net/</url>.</p

Characterization of relationships between transcriptional units and operon structures in Bacillus subtilis and Escherichia coli

BACKGROUND: Operon structures play an important role in transcriptional regulation in prokaryotes. However, there have been fewer studies on complicated operon structures in which the transcriptional units vary with changing environmental conditions. Information about such complicated operons is helpful for predicting and analyzing operon structures, as well as understanding gene functions and transcriptional regulation. RESULTS: We systematically analyzed the experimentally verified transcriptional units (TUs) in Bacillus subtilis and Escherichia coli obtained from ODB and RegulonDB. To understand the relationships between TUs and operons, we defined a new classification system for adjacent gene pairs, divided into three groups according to the level of gene co-regulation: operon pairs (OP) belong to the same TU, sub-operon pairs (SOP) that are at the transcriptional boundaries within an operon, and non-operon pairs (NOP) belonging to different operons. Consequently, we found that the levels of gene co-regulation was correlated to intergenic distances and gene expression levels. Additional analysis revealed that they were also correlated to the levels of conservation across about 200 prokaryotic genomes. Most interestingly, we found that functional associations in SOPs were more observed in the environmental and genetic information processes. CONCLUSION: Complicated operon strucutures were correlated with genome organization and gene expression profiles. Such intricately regulated operons allow functional differences depending on environmental conditions. These regulatory mechanisms are helpful in accommodating the variety of changes that happen around the cell. In addition, such differences may play an important role in the evolution of gene order across genomes

Regulation of chromosomal replication initiation by oriC-proximal DnaA-box clusters in Bacillus subtilis

Bacterial chromosome replication is initiated by binding of DnaA to a DnaA-box cluster (DBC) within the replication origin (oriC). In Bacillus subtilis, six additional DBCs are found outside of oriC and some are known to be involved in transcriptional regulation of neighboring genes. A deletion mutant lacking the six DBCs (Δ6) initiated replication early. Further, inactivation of spo0J in Δ6 cells yielded a pleiotropic phenotype, accompanied by severe growth inhibition. However, a spontaneous suppressor in soj or a deletion of soj, which stimulates DnaA activity in the absence of Spo0J, counteracted these effects. Such abnormal phenotypic features were not observed in a mutant background in which replication initiation was driven by a plasmid-derived replication origin. Moreover, introduction of a single DBC at various ectopic positions within the Δ6 chromosome partly suppressed the early-initiation phenotype, but this was dependent on insertion location. We propose that DBCs negatively regulate replication initiation by interacting with DnaA molecules and play a major role, together with Spo0J/Soj, in regulating the activity of DnaA

Predicting state transitions in the transcriptome and metabolome using a linear dynamical system model

<p>Abstract</p> <p>Background</p> <p>Modelling of time series data should not be an approximation of input data profiles, but rather be able to detect and evaluate dynamical changes in the time series data. Objective criteria that can be used to evaluate dynamical changes in data are therefore important to filter experimental noise and to enable extraction of unexpected, biologically important information.</p> <p>Results</p> <p>Here we demonstrate the effectiveness of a Markov model, named the Linear Dynamical System, to simulate the dynamics of a transcript or metabolite time series, and propose a probabilistic index that enables detection of time-sensitive changes. This method was applied to time series datasets from <it>Bacillus subtilis </it>and <it>Arabidopsis thaliana </it>grown under stress conditions; in the former, only gene expression was studied, whereas in the latter, both gene expression and metabolite accumulation. Our method not only identified well-known changes in gene expression and metabolite accumulation, but also detected novel changes that are likely to be responsible for each stress response condition.</p> <p>Conclusion</p> <p>This general approach can be applied to any time-series data profile from which one wishes to identify elements responsible for state transitions, such as rapid environmental adaptation by an organism.</p

High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq

Accurate identification of the DNA-binding sites of transcription factors and other DNA-binding proteins on the genome is crucial to understanding their molecular interactions with DNA. Here, we describe a new method: Genome Footprinting by high-throughput sequencing (GeF-seq), which combines in vivo DNase I digestion of genomic DNA with ChIP coupled with high-throughput sequencing. We have determined the in vivo binding sites of a Bacillus subtilis global regulator, AbrB, using GeF-seq. This method shows that exact DNA-binding sequences, which were protected from in vivo DNase I digestion, were resolved at a comparable resolution to that achieved by in vitro DNase I footprinting, and this was simply attained without the necessity of prediction by peak-calling programs. Moreover, DNase I digestion of the bacterial nucleoid resolved the closely positioned AbrB-binding sites, which had previously appeared as one peak in ChAP-chip and ChAP-seq experiments. The high-resolution determination of AbrB-binding sites using GeF-seq enabled us to identify bipartite TGGNA motifs in 96% of the AbrB-binding sites. Interestingly, in a thousand binding sites with very low-binding intensities, single TGGNA motifs were also identified. Thus, GeF-seq is a powerful method to elucidate the molecular mechanism of target protein binding to its cognate DNA sequences

Distribution of Stable DnaA-Binding Sites on the Bacillus Subtilis Genome Detected using a Modified ChIP-chip Method

We developed a modified ChIP-chip method, designated ChAP-chip (Chromatin Affinity Precipitation coupled with tiling chip). The binding sites of Bacillus subtilis Spo0J determined using this technique were consistent with previous findings. A DNA replication initiator protein, DnaA, formed stable complexes at eight intergenic regions on the B. subtilis genome. Characterization of the binding sequences suggested that two factors—the local density of DnaA boxes and their affinities for DnaA—are critical for stable binding. We further showed that in addition to autoregulation, DnaA directly modulate the expression of sda in a positive, and ywlC and yydA in a negative manner. Examination of possible stable DnaA-binding sequences in other Bacillus species suggested that DnaA-dependent regulation of those genes is maintained in most bacteria examined, supporting their biological significance. In addition, a possible stable DnaA-binding site downstream of gcp is also suggested to be conserved. Furthermore, potential DnaA-binding sequences specific for each bacterium have been identified, generally in close proximity to oriC. These findings suggest that DnaA plays several additional roles, such as control of the level of effective initiator, ATP-DnaA, and/or stabilization of the domain structure of the genome around oriC for the proper initiation of chromosome replication

The entire organization of transcription units on the Bacillus subtilis genome

<p>Abstract</p> <p>Background</p> <p>In the post-genomic era, comprehension of cellular processes and systems requires global and non-targeted approaches to handle vast amounts of biological information.</p> <p>Results</p> <p>The present study predicts transcription units (TUs) in <it>Bacillus subtilis</it>, based on an integrated approach involving DNA sequence and transcriptome analyses. First, co-expressed gene clusters are predicted by calculating the Pearson correlation coefficients of adjacent genes for all the genes in a series that are transcribed in the same direction with no intervening gene transcribed in the opposite direction. Transcription factor (TF) binding sites are then predicted by detecting statistically significant TF binding sequences on the genome using a position weight matrix. This matrix is a convenient way to identify sites that are more highly conserved than others in the entire genome because any sequence that differs from a consensus sequence has a lower score. We identify genes regulated by each of the TFs by comparing gene expression between wild-type and TF mutants using a one-sided test. By applying the integrated approach to 11 σ factors and 17 TFs of <it>B. subtilis</it>, we are able to identify fewer candidates for genes regulated by the TFs than were identified using any single approach, and also detect the known TUs efficiently.</p> <p>Conclusion</p> <p>This integrated approach is, therefore, an efficient tool for narrowing searches for candidate genes regulated by TFs, identifying TUs, and estimating roles of the σ factors and TFs in cellular processes and functions of genes composing the TUs.</p

Enhanced Recombinant Protein Productivity by Genome Reduction in Bacillus subtilis

The emerging field of synthetic genomics is expected to facilitate the generation of microorganisms with the potential to achieve a sustainable society. One approach towards this goal is the reduction of microbial genomes by rationally designed deletions to create simplified cells with predictable behavior that act as a platform to build in various genetic systems for specific purposes. We report a novel Bacillus subtilis strain, MBG874, depleted of 874 kb (20%) of the genomic sequence. When compared with wild-type cells, the regulatory network of gene expression of the mutant strain is reorganized after entry into the transition state due to the synergistic effect of multiple deletions, and productivity of extracellular cellulase and protease from transformed plasmids harboring the corresponding genes is remarkably enhanced. To our knowledge, this is the first report demonstrating that genome reduction actually contributes to the creation of bacterial cells with a practical application in industry. Further systematic analysis of changes in the transcriptional regulatory network of MGB874 cells in relation to protein productivity should facilitate the generation of improved B. subtilis cells as hosts of industrial protein production