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Bacillus subtilis lacking the DnaJK chaperone has not been reported to exhibit a distinct phenotype. However, our study revealed proline-dependent growth in a minimal medium in the dnaJ ::Tn strain. Inhibition of spoIVCA expression in this strain was identified as a probable cause of the sporulation deficiency in previous and current studies using a single cell-level analysis. We also observed posttranscriptional regulation of proBA by the DnaJK and YlxR(RnpM)/RNase P complex. LacZ analyses of proB::lacZ in different backgrounds suggested that the above regulation ultimately functions in mRNA metabolism. In DnaJK-deficient cells, the nascent peptide may be misfolded, and if DnaJK chaperone activity is lost, such a signal may be transferred to RNase P. Therefore, proBA mRNA may be degraded in an RNase P-dependent manner if the misfolding of the polypeptide translated from this mRNA is detected. This system is useful for reducing the biological costs of futile mRNA elongation.

Objective We observed that the addition of glucose enhanced the expression of sigX and sigM, encoding extra-cytoplasmic function sigma factors in Bacillus subtilis . Several regulatory factors were identified for this phenomenon, including YqfO, CshA (RNA helicase), and YlxR (nucleoid-associated protein). Subsequently, the relationships among these regulators were analyzed. Among them, YqfO is conserved in many bacterial genomes and may function as a metal ion insertase or metal chaperone, but has been poorly characterized. Thus, to further characterize YqfO, we performed RNA sequencing (RNA-seq) analysis of YqfO in addition to CshA and YlxR. Results We first performed comparative RNA-seq to detect the glucose-responsive genes. Next, to determine the regulatory effects of YqfO in addition to CshA and YlxR, three pairs of comparative RNA-seq analyses were performed ( yqfO /wt, cshA /wt, and ylxR /wt). We observed relatively large regulons (approximately 420, 780, and 180 for YqfO, CshA, and YlxR, respectively) and significant overlaps, indicating close relationships among the three regulators. This study is the first to reveal that YqfO functions as a global regulator in B. subtilis .

Bacteria have developed various motility mechanisms to adapt to a variety of solid surfaces. A rhizosphere isolate, Paenibacillus sp. NAIST15-1, exhibited unusual motility behavior. When spotted onto 1.5% agar media, Paenibacillus sp. formed many colonies, each of which moved around actively at a speed of 3.6 μm/sec. As their density increased, each moving colony began to spiral, finally forming a static round colony. Despite its unusual motility behavior, draft genome sequencing revealed that both the composition and organization of flagellar genes in Paenibacillus sp. were very similar to those in Bacillus subtilis. Disruption of flagellar genes and flagellar stator operons resulted in loss of motility. Paenibacillus sp. showed increased transcription of flagellar genes and hyperflagellation on hard agar media. Thus, increased flagella and their rotation drive Paenibacillus sp. motility. We also identified a large extracellular protein, CmoA, which is conserved only in several Paenibacillus and related species. A cmoA mutant could neither form moving colonies nor move on hard agar media; however, motility was restored by exogenous CmoA. CmoA was located around cells and enveloped cell clusters. Comparison of cellular behavior between the wild type and cmoA mutant indicated that extracellular CmoA is involved in drawing water out of agar media and/or smoothing the cell surface interface. This function of CmoA probably enables Paenibacillus sp. to move on hard agar media.

Fpr1 (FK506-sensitive proline rotamase 1), a protein of the FKBP12 (FK506-binding protein 12 kDa) family in Saccharomyces cerevisiae, is a primary target for the immunosuppressive agents FK506 and rapamycin. Fpr1 inhibits calcineurin and TORC1 (target of rapamycin complex 1) when bound to FK506 and rapamycin, respectively. Although Fpr1 is recognised to play a crucial role in the efficacy of these drugs, its physiological functions remain unclear. In a hmo1[DELTA] (high mobility group family 1-deleted) yeast strain, deletion of FPR1 induced severe growth defects, which could be alleviated by increasing the copy number of RPL25 (ribosome protein of the large subunit 25), suggesting that RPL25 expression was affected in hmo1[DELTA]fpr1[DELTA] cells. In the current study, extensive chromatin immunoprecipitation (ChIP) and ChIP-sequencing analyses revealed that Fpr1 associates specifically with the upstream activating sequences of nearly all RPG (ribosomal protein gene) promoters, presumably in a manner dependent on Rap1 (repressor/activator site binding protein 1). Intriguingly, Fpr1 promotes the binding of Fhl1/Ifh1 (forkhead-like 1/interacts with forkhead 1), two key regulators of RPG transcription, to certain RPG promoters independently of and/or cooperatively with Hmo1. Furthermore, mutation analyses of Fpr1 indicated that for transcriptional function on RPG promoters, Fpr1 requires its N-terminal domain and the binding surface for rapamycin, but not peptidyl-prolyl isomerase activity. Notably, Fpr1 orthologues from other species also inhibit TORC1 when bound to rapamycin, but do not regulate transcription in yeast, which suggests that these two functions of Fpr1 are independent of each other.

Microalgae such as cyanobacteria convert CO2 to compatible drop‐in fuels, such as alkanes. However, the production yield is approximately 0.05–1.0% of the dry weight of natural algae. Here, we aimed to study the role of transcriptional expression, mRNA molecular structure and culture‐dependent accumulation of alkanes from two cyanobacteria species. The transcription start sites of the alkane biosynthesis genes ado and aar were identified in the representative cyanobacteria strains Synechocystis sp. PCC 6803 and Limnothrix sp. SK1‐2‐1, which produce heptadecane and pentadecane, respectively. This characterisation revealed the potential promoters and unique mRNA structures of the ado and aar genes in these species. Transcripts from these genes were induced more in the nitrogen‐depleted BG11 (BG11‐N) culture than in the BG11 culture, although the biomass was reduced, and as such the amount of alkanes obtained per unit medium was greater for BG11 than for BG11‐N. PCC 6803 transconjugants carrying alkane biosynthesis genes from PCC 6803 or SK1‐2‐1 showed an approximately 1.8‐ to 2.3‐fold increase in heptadecane production compared to the control strain when grown on BG11 cultures without any nitrogen depletion. These results suggest that not only the enzymes ADO/AAR but also the intracellular production of fatty acyl‐ACP substrates may be important for the mass production of target alkanes. The transcription and mRNA structures of the alkane biosynthesis genes ado/aar were analysed in the representative cyanobacteria PCC 6803 and SK1‐2‐1, which produce C17H36 and C15H32, respectively. PCC 6803 transconjugants carrying these genes showed a 1.8‐ to 2.3‐fold increase in C17H36 production. The results indicate that ADO/AAR activities, together with fatty acyl‐ACP mass, are important to produce target alkanes.

Acid-resistance systems are essential for pathogenic Escherichia coli to survive in the strongly acidic environment of the human stomach (pH < 2.5). Among these, the glutamic acid decarboxylase (GAD) system is the most effective. However, the precise mechanism of GAD induction is unknown. We previously reported that a tolC mutant lacking the TolC outer membrane channel was defective in GAD induction. Here, we show that indole, a substrate of TolC-dependent efflux pumps and produced by the tryptophanase encoded by the tnaA gene, negatively regulates GAD expression. GAD expression was restored by deleting tnaA in the tolC mutant; in wild-type E. coli , it was suppressed by adding indole to the growth medium. RNA-sequencing revealed that tnaA mRNA levels drastically decreased upon exposure to moderately acidic conditions (pH 5.5). This decrease was suppressed by RNase E deficiency. Collectively, our results demonstrate that the RNase E-dependent degradation of tnaA mRNA is accelerated upon acid exposure, which decreases intracellular indole concentrations and triggers GAD induction.

Erythrobacter flavus strain KJ5 (formerly called Erythrobacter sp. strain KJ5) is a yellowish marine bacterium that was isolated from a hard coral Acropora nasuta in the Karimunjawa Islands, Indonesia. The complete genome sequence of the bacterium has been reported recently. In this study, we examined the carotenoid composition of this bacterium using high-performance liquid chromatography coupled with ESI-MS/MS. We found that the bacterium produced sulfur-containing carotenoids, i.e., caloxanthin sulfate and nostoxanthin sulfate, as the most abundant carotenoids. A new carotenoid zeaxanthin sulfate was detected based on its ESI-MS/MS spectrum. The unique presence of sulfated carotenoids found among the currently known species of the Erythrobacter genus were discussed.

Unlike bacteria such as Escherichia coli and Bacillus subtilis, several species of freshwater cyanobacteria are known to contain multiple chromosomal copies per cell, at all stages of their cell cycle. We have characterized the replication of multi-copy chromosomes in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942). In Synechococcus 7942, the replication of multi-copy chromosome is asynchronous, not only among cells but also among multi-copy chromosomes. This suggests that DNA replication is not tightly coupled to cell division in Synechococcus 7942. To address this hypothesis, we analysed the relationship between DNA replication and cell doubling at various growth phases of Synechococcus 7942 cell culture. Three distinct growth phases were characterised in Synechococcus 7942 batch culture: lag phase, exponential phase, and arithmetic (linear) phase. The chromosomal copy number was significantly higher during the lag phase than during the exponential and linear phases. Likewise, DNA replication activity was higher in the lag phase cells than in the exponential and linear phase cells, and the lag phase cells were more sensitive to nalidixic acid, a DNA gyrase inhibitor, than cells in other growth phases. To elucidate physiological differences in Synechococcus 7942 during the lag phase, we analysed the metabolome at each growth phase. In addition, we assessed the accumulation of central carbon metabolites, amino acids, and DNA precursors at each phase. The results of these analyses suggest that Synechococcus 7942 cells prepare for cell division during the lag phase by initiating intensive chromosomal DNA replication and accumulating metabolites necessary for the subsequent cell division and elongation steps that occur during the exponential growth and linear phases.

PmlR2, a class II LitR/CarH family transcriptional regulator, and PmSB-LOV, a “short” LOV-type blue light photoreceptor, are adjacently encoded in Pseudomonas mendocina NBRC 14162. An effector protein for the “short” LOV-type photoreceptor in Pseudomonas has not yet been identified. Here, we show that PmlR2 is an effector protein of PmSB-LOV. Transcriptional analyses revealed that the expression of genes located near pmlR2 and its homolog gene, pmlR1, was induced in response to illumination. In vitro DNA–protein binding analyses showed that recombinant PmlR2 directly binds to the promoter region of light-inducible genes. Furthermore PmSB-LOV exhibited a typical LOV-type light-induced spectral change. Gel-filtration chromatography demonstrated that the illuminated PmSB-LOV was directly associated with PmlR2, whereas non-illuminated proteins did not interact. The inhibition of PmlR2 function following PmSB-LOV binding was verified by surface plasmon resonance: the DNA-binding ability of PmlR2 was specifically inhibited in the presence of blue light-illuminated-PmSB-LOV. An In vitro transcription assay showed a dose-dependent reduction in PmlR2 repressor activity in the presence of illuminated PmSB-LOV. Overall, evidence suggests that the DNA-binding activity of PmlR2 is inhibited by its direct association with blue light-activated PmSB-LOV, enabling transcription of light-inducible promoters by RNA polymerase.

Expression of genes encoding NAPs is often temporally regulated. According to results from single-cell analysis, the ylxR gene is induced by glucose and expressed in a bistable mode. These characteristics have not previously been reported for NAP gene expression. Transcriptional profiling of the ylxR disruptant revealed a change in the expression levels of approximately 400 genes, including genes for synthesis of 12 amino acids and 4 nucleotides, in addition to the SigX/M regulons. Thus, YlxR is a critical regulator of glucose response in B. subtilis . Glucose is the most favorable carbon source for the majority of bacteria, which have several glucose-responsive gene networks. Recently, we found that in Bacillus subtilis , glucose induces expression of the extracellular sigma factor genes sigX / M . To explore the factors affecting this phenomenon, we performed a transposon mutagenesis screen for mutants with no glucose induction (GI) of sigX-lacZ and identified ylxR . YlxR is widely conserved in eubacteria. Further analysis revealed that ylxR is induced by glucose addition. In vitro DNA-binding and cytological studies suggested that YlxR is a nucleoid-associated protein (NAP) in B. subtilis . In many cases, NAPs influence transcription, recombination, and genome stability. Thus, we performed transcriptome sequencing (RNA-Seq) analysis to evaluate the impact of ylxR disruption on the transcriptome in the presence of glucose and observed that YlxR has a profound impact on metabolic gene expression in addition to that of four sigma factor genes. The wide fluctuations of gene expression may result in abolition of GI of sigX / M in the ylxR disruptant. IMPORTANCE Expression of genes encoding NAPs is often temporally regulated. According to results from single-cell analysis, the ylxR gene is induced by glucose and expressed in a bistable mode. These characteristics have not previously been reported for NAP gene expression. Transcriptional profiling of the ylxR disruptant revealed a change in the expression levels of approximately 400 genes, including genes for synthesis of 12 amino acids and 4 nucleotides, in addition to the SigX/M regulons. Thus, YlxR is a critical regulator of glucose response in B. subtilis .