Complete genome sequence of Paraburkholderia sp. strain 22B1P capable of utilizing 3-chlorobenzoate as a carbon source.

Paraburkholderia sp. strain 22B1P utilizes 3-chlorobenzoate as a carbon source. Complete genome sequencing of strain 22B1P revealed two chromosomes and two plasmids. The genes involved in the conversion of 3-chlorobenzoate to 3-chlorocatechol and those involved in the conversion of 3-chlorocatechol to 3-oxoadipate were located on chromosomes 2 and 1, respectively.

Implication of amino acid metabolism and cell surface integrity for the thermotolerance mechanism in the thermally adapted acetic acid bacterium Acetobacter pasteurianus TH-3.

IMPORTANCE: Acetobacter pasteurianus, an industrial vinegar-producing strain, is suffered by fermentation stress such as fermentation heat and/or high concentrations of acetic acid. By an experimental evolution approach, we have obtained a stress-tolerant strain, exhibiting significantly increased growth and acetic acid fermentation ability at higher temperatures. In this study, we report that only the three gene mutations of ones accumulated during the adaptation process, ansP, dctD, and glnD, were sufficient to reproduce the increased thermotolerance of A. pasteurianus. These mutations resulted in cell envelope modification, including increased phospholipid and lipopolysaccharide synthesis, increased respiratory activity, and cell size reduction. The phenotypic changes may cooperatively work to make the adapted cell thermotolerant by enhancing cell surface integrity, nutrient or oxygen availability, and energy generation.

yaaJ, the tRNA-Specific Adenosine Deaminase, Is Dispensable in Bacillus subtilis.

Post-transcriptional modifications of tRNA are crucial for their core function. The inosine (I; 6-deaminated adenosine) at the first position in the anticodon of tRNAArg(ICG) modulates the decoding capability and is generally considered essential for reading CGU, CGC, and CGA codons in eubacteria. We report here that the Bacillus subtilis yaaJ gene encodes tRNA-specific adenosine deaminase and is non-essential for viability. A ß-galactosidase reporter assay revealed that the translational activity of CGN codons was not impaired in the yaaJ-deletion mutant. Furthermore, tRNAArg(CCG) responsible for decoding the CGG codon was dispensable, even in the presence or absence of yaaJ. These results strongly suggest that tRNAArg with either the anticodon ICG or ACG has an intrinsic ability to recognize all four CGN codons, providing a fundamental concept of non-canonical wobbling mediated by adenosine and inosine nucleotides in the anticodon. This is the first example of the four-way wobbling by inosine nucleotide in bacterial cells. On the other hand, the absence of inosine modification induced +1 frameshifting, especially at the CGA codon. Additionally, the yaaJ deletion affected growth and competency. Therefore, the inosine modification is beneficial for translational fidelity and proper growth-phase control, and that is why yaaJ has been actually conserved in B. subtilis.

A high-temperature sensitivity of Synechococcus elongatus PCC 7942 due to a tRNA-Leu mutation.

Certain mutations of the model cyanobacterium Synechococcus elongatus PCC 7942 during laboratory storage have resulted in some divergent phenotypes. One laboratory-stored strain (H1) shows a temperature-sensitive (ts) growth phenotype at 40 °C. Here, we investigated the reason for this temperature sensitivity. Whole genome sequencing of H1 identified a single nucleotide mutation in synpcc7942_R0040 encoding tRNA-Leu(CAA). The mutation decreases the length of the tRNA-Leu t-arm from 5 to 4 base pairs, and this explains the ts phenotype. Secondary mutations suppressing the ts phenotype were identified in synpcc7942_1640, which putatively encodes a NYN domain-containing protein (nynA). The NYN domain is thought to be involved in tRNA/rRNA degradation. Thus, the structural stability of tRNA-Leu is critical for growth at 40 °C in Synechococcus elongatus PCC 7942.

Class II LitR serves as an effector of "short" LOV-type blue-light photoreceptor in Pseudomonas mendocina.

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.

Possible involvement of extracellular polymeric substrates of Antarctic cyanobacterium Nostoc sp. strain SO-36 in adaptation to harsh environments.

Cyanobacteria are some of the primary producers in extremely cold biospheres such as the Arctic, Antarctic, and vast ice sheets. Many genera of cyanobacteria are identified from these harsh environments, but their specific mechanisms for cold adaptation are not fully understood. Nostoc sp. strain SO-36 is a cyanobacterium isolated in Antarctica more than 30 years ago and regarded as a psychrotolelant species. To determine whether the strain is psychrotolelant or psychrophilic, it was first grown at 30 °C and 10 °C. The cells grew exponentially at 30 °C, but their growth stopped at 10 °C, indicating that the strain is only psychrotolerant. Microscopic analysis revealed that the morphology of the cells grown at 30 °C was filamentous and differentiated heterocysts, which are specialized cells for gaseous nitrogen fixation under nitrogen-deprived conditions, indicating that the strain can grow diazotrophically. The cells grown at 10 °C have a smaller size, shortened filament length and decreased chlorophyll content per cell. At 10 °C, the cells are aggregated with extracellular polymeric substrates (EPSs), which is a common mechanism to protect cells from ultraviolet light. These results imply that segmentation into short filaments was induced by photodamage at low temperatures. To fully understand the adaptation mechanisms of Nostoc sp. strain SO-36 for low-temperature conditions, next-generation sequencing analyses were conducted. Complete genome sequence of the strain revealed that it has one main chromosome of approximately 6.8 Mbp with 4 plasmids, including 6855 coding sequences, 48 tRNA genes, 4 copies of rRNA operons, and 5 CRISPR regions. Putative genes for EPS biosynthesis were found to be conserved in Nostocaceae regardless of their habitat. These results provide basic information to understand the adaptation mechanisms at low temperatures, and the strain can be a model organism to analyze adaptation to extreme environments.

Mutations in degP and spoT Genes Mediate Response to Fermentation Stress in Thermally Adapted Strains of Acetic Acid Bacterium Komagataeibacter medellinensis NBRC 3288.

An acetic acid bacterium, Komagataeibacter medellinensis NBRC 3288, was adapted to higher growth temperatures through an experimental evolution approach in acetic acid fermentation conditions, in which the cells grew under high concentrations of ethanol and acetic acid. The thermally adapted strains were shown to exhibit significantly increased growth and fermentation ability, compared to the wild strain, at higher temperatures. Although the wild cells were largely elongated and exhibited a rough cell surface, the adapted strains repressed the elongation and exhibited a smaller cell size and a smoother cell surface than the wild strain. Among the adapted strains, the ITO-1 strain isolated during the initial rounds of adaptation was shown to have three indel mutations in the genes gyrB, degP, and spoT. Among these, two dispensable genes, degP and spoT, were further examined in this study. Rough cell surface morphology related to degP mutation suggested that membrane vesicle-like structures were increased on the cell surface of the wild-type strain but repressed in the ITO-1 strain under high-temperature acetic acid fermentation conditions. The ΔdegP strain could not grow at higher temperatures and accumulated a large amount of membrane vesicles in the culture supernatant when grown even at 30°C, suggesting that the degP mutation is involved in cell surface stability. As the spoT gene of ITO-1 lost a 3'-end of 424 bp, which includes one (Act-4) of the possible two regulatory domains (TGS and Act-4), two spoT mutant strains were created: one (ΔTGSAct) with a drug cassette in between the 5'-half catalytic domain and 3'-half regulatory domains of the gene, and the other (ΔAct-4) in between TGS and Act-4 domains of the regulatory domain. These spoT mutants exhibited different growth responses; ΔTGSAct grew better in both the fermentation and non-fermentation conditions, whereas ΔAct-4 did only under fermentation conditions, such as ITO-1 at higher temperatures. We suggest that cell elongation and/or cell size are largely related to these spoT mutations, which may be involved in fermentation stress and thermotolerance.

Evolutionary Adaptation by Repetitive Long-Term Cultivation with Gradual Increase in Temperature for Acquiring Multi-Stress Tolerance and High Ethanol Productivity in Kluyveromyces marxianus DMKU 3-1042.

During ethanol fermentation, yeast cells are exposed to various stresses that have negative effects on cell growth, cell survival, and fermentation ability. This study, therefore, aims to develop Kluyveromyces marxianus-adapted strains that are multi-stress tolerant and to increase ethanol production at high temperatures through a novel evolutionary adaptation procedure. K. marxianus DMKU 3-1042 was subjected to repetitive long-term cultivation with gradual increases in temperature (RLCGT), which exposed cells to various stresses, including high temperatures. In each cultivation step, 1% of the previous culture was inoculated into a medium containing 1% yeast extract, 2% peptone, and 2% glucose, and cultivation was performed under a shaking condition. Four adapted strains showed increased tolerance to ethanol, furfural, hydroxymethylfurfural, and vanillin, and they also showed higher production of ethanol in a medium containing 16% glucose at high temperatures. One showed stronger ethanol tolerance. Others had similar phenotypes, including acetic acid tolerance, though genome analysis revealed that they had different mutations. Based on genome and transcriptome analyses, we discuss possible mechanisms of stress tolerance in adapted strains. All adapted strains gained a useful capacity for ethanol fermentation at high temperatures and improved tolerance to multi-stress. This suggests that RLCGT is a simple and efficient procedure for the development of robust strains.

Draft Genome Sequence of Lactiplantibacillus plantarum NMZ-1139, Isolated from Whisky Mash.

Lactiplantibacillus plantarum NMZ-1139 was isolated from whisky mash and applied to sour beer production. Here, we report the draft genome sequence of L. plantarum NMZ-1139, which contains 3,117 protein-coding sequences, including genes associated with hop resistance, such as horA and hitA.

RNA-seq analysis identified glucose-responsive genes and YqfO as a global regulator in Bacillus subtilis.

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.

Genome sequencing of the NIES Cyanobacteria collection with a focus on the heterocyst-forming clade.

Cyanobacteria are a diverse group of Gram-negative prokaryotes that perform oxygenic photosynthesis. Cyanobacteria have been used for research on photosynthesis and have attracted attention as a platform for biomaterial/biofuel production. Cyanobacteria are also present in almost all habitats on Earth and have extensive impacts on global ecosystems. Given their biological, economical, and ecological importance, the number of high-quality genome sequences for Cyanobacteria strains is limited. Here, we performed genome sequencing of Cyanobacteria strains in the National Institute for Environmental Studies microbial culture collection in Japan. We sequenced 28 strains that can form a heterocyst, a morphologically distinct cell that is specialized for fixing nitrogen, and 3 non-heterocystous strains. Using Illumina sequencing of paired-end and mate-pair libraries with in silico finishing, we constructed highly contiguous assemblies. We determined the phylogenetic relationship of the sequenced genome assemblies and found potential difficulties in the classification of certain heterocystous clades based on morphological observation. We also revealed a bias on the sequenced strains by the phylogenetic analysis of the 16S rRNA gene including unsequenced strains. Genome sequencing of Cyanobacteria strains deposited in worldwide culture collections will contribute to understanding the enormous genetic and phenotypic diversity within the phylum Cyanobacteria.

Physiological and genomic analysis of newly-isolated polysaccharide synthesizing cyanobacterium Chroococcus sp. FPU101 and chemical analysis of the exopolysaccharide.

A unicellular cyanobacterium that produces a large amount of exopolysaccharide (EPS) was isolated. The isolate, named Chroococcus sp. FPU101, grew between 20 and 30°C and at light intensities between 10 and 80 µmol m-2 s-1. Purified EPS from Chroococcus sp. FPU101 had a molecular size of 5.9 × 103 kDa and contained galactose, rhamnose, fucose, xylose, mannose, glucose, galacturonic acid, and glucuronic acid at a molar ratio of 17.2:15.9:14.1:11.0:9.6:9.5:13.0:9.7. The EPS content significantly increased when the NaCl concentration in the medium was increased from 1.7 to 100 mM. However, high NaCl concentrations did not significantly affect the molecular size or chemical composition of the EPS. The genes wza, wzb, wzc, wzx, wzy, and wzz that are involved in EPS synthesis were conserved in the genome of Chroococcus sp. FPU101, which was sequenced in this study. These results suggest that the Wzy-dependent pathway is potentially involved in EPS production in this organism.

Identification of Transcription Factors and the Regulatory Genes Involved in Triacylglycerol Accumulation in the Unicellular Red Alga Cyanidioschyzon merolae.

Microalgal triacylglycerols (TAGs) are a good feedstock for liquid biofuel production. Improving the expression and/or function of transcription factors (TFs) involved in TAG accumulation may increase TAG content; however, information on microalgae is still lacking. In this study, 14 TFs in the unicellular red alga Cyanidioschyzon merolae were identified as candidate TFs regulating TAG accumulation using available transcriptome and phosphoproteome data under conditions driving TAG accumulation. To investigate the roles of these TFs, we constructed TF-overexpression strains and analyzed lipid droplet (LD) formation and TAG contents in the cells grown under standard conditions. Based on the results, we identified four TFs involved in LD and TAG accumulation. RNA-Seq analyses were performed to identify genes regulated by the four TFs using each overexpression strain. Among the TAG biosynthesis-related genes, only the gene encoding the endoplasmic reticulum-localized lysophosphatidic acid acyltransferase 1 (LPAT1) was notably increased among the overexpression strains. In the LPAT1 overexpression strain, TAG accumulation was significantly increased compared with the control strain under normal growth conditions. These results indicate that the four TFs positively regulate TAG accumulation by changing their target gene expression in C. merolae.

Transcriptome differences between Cupriavidus necator NH9 grown with 3-chlorobenzoate and that grown with benzoate.

RNA-seq analysis of Cupriavidus necator NH9, a 3-chlorobenzoate degradative bacterium, cultured with 3-chlorobenzaote and benzoate, revealed strong induction of genes encoding enzymes in degradation pathways of the respective compound, including the genes to convert 3-chlorobenzaote and benzoate to chlorocatechol and catechol, respectively, and the genes of chlorocatechol ortho-cleavage pathway for conversion to central metabolites. The genes encoding transporters, components of the stress response, flagellar proteins, and chemotaxis proteins showed altered expression patterns between 3-chlorobenzoate and benzoate. Gene Ontology enrichment analysis revealed that chemotaxis-related terms were significantly upregulated by benzoate compared with 3-chlorobenzoate. Consistent with this, in semisolid agar plate assays, NH9 cells showed stronger chemotaxis to benzoate than to 3-chlorobenzoate. These results, combined with the absence of genes related to uptake/chemotaxis for 3-chlorobenzoate located closely to the degradation genes of 3-chlorobenzoate, suggested that NH9 has not fully adapted to the utilization of chlorinated benzoate, unlike benzoate, in nature.

Complete sequence and structure of the genome of the harmful algal bloom-forming cyanobacterium Planktothrix agardhii NIES-204T and detailed analysis of secondary metabolite gene clusters.

Planktothrix species are distributed worldwide, and these prevalent cyanobacteria occasionally form potentially devastating toxic blooms. Given the ecological and taxonomic importance of Planktothrix agardhii as a bloom species, we set out to determine the complete genome sequence of the type strain Planktothrix agardhii NIES-204. Remarkably, we found that the 5S ribosomal RNA genes are not adjacent to the 16S and 23S ribosomal RNA genes. The genomic structure of P. agardhii NIES-204 is highly similar to that of another P. agardhii strain isolated from a geographically distant site, although they differ distinctly by a large inversion. We identified numerous gene clusters that encode the components of the metabolic pathways that generate secondary metabolites. We found that the aeruginosin biosynthetic gene cluster was more similar to that of another toxic bloom-forming cyanobacterium Microcystis aeruginosa than to that of other strains of Planktothrix, suggesting horizontal gene transfer. Prenyltransferases encoded in the prenylagaramide gene cluster of Planktothrix strains were classified into two phylogenetically distinct types, suggesting a functional difference. In addition to the secondary metabolite gene clusters, we identified genes for inorganic nitrogen and phosphate uptake components and gas vesicles. Our findings contribute to further understanding of the ecologically important genus Planktothrix.

Reacquisition of light-harvesting genes in a marine cyanobacterium confers a broader solar niche.

The evolution of phenotypic plasticity, i.e., the environmental induction of alternative phenotypes by the same genotype, can be an important mechanism of biological diversification.1,2 For example, an evolved increase in plasticity may promote ecological niche expansion as well as the innovation of novel traits;3 however, both the role of phenotypic plasticity in adaptive evolution and its underlying mechanisms are still poorly understood.4,5 Here, we report that the Chlorophyll d-producing marine cyanobacterium Acaryochloris marina strain MBIC11017 has evolved greater photosynthetic plasticity by reacquiring light-harvesting genes via horizontal gene transfer. The genes, which had been lost by the A. marina ancestor, are involved in the production and degradation of the light-harvesting phycobiliprotein phycocyanin. A. marina MBIC11017 exhibits a high degree of wavelength-dependence in phycocyanin production, and this ability enables it to grow with yellow and green light wavelengths that are inaccessible to other A. marina. Consequently, this strain has a broader solar niche than its close relatives. We discuss the role of horizontal gene transfer for regaining a lost phenotype in light of Dollo's Law6 that the loss of a complex trait is irreversible.

The Rubisco small subunits in the green algal genus Chloromonas provide insights into evolutionary loss of the eukaryotic carbon-concentrating organelle, the pyrenoid.

BACKGROUND: Pyrenoids are protein microcompartments composed mainly of Rubisco that are localized in the chloroplasts of many photosynthetic organisms. Pyrenoids contribute to the CO2-concentrating mechanism. This organelle has been lost many times during algal/plant evolution, including with the origin of land plants. The molecular basis of the evolutionary loss of pyrenoids is a major topic in evolutionary biology. Recently, it was hypothesized that pyrenoid formation is controlled by the hydrophobicity of the two helices on the surface of the Rubisco small subunit (RBCS), but the relationship between hydrophobicity and pyrenoid loss during the evolution of closely related algal/plant lineages has not been examined. Here, we focused on, the Reticulata group of the unicellular green algal genus Chloromonas, within which pyrenoids are present in some species, although they are absent in the closely related species. RESULTS: Based on de novo transcriptome analysis and Sanger sequencing of cloned reverse transcription-polymerase chain reaction products, rbcS sequences were determined from 11 strains of two pyrenoid-lacking and three pyrenoid-containing species of the Reticulata group. We found that the hydrophobicity of the RBCS helices was roughly correlated with the presence or absence of pyrenoids within the Reticulata group and that a decrease in the hydrophobicity of the RBCS helices may have primarily caused pyrenoid loss during the evolution of this group. CONCLUSIONS: Although we suggest that the observed correlation may only exist for the Reticulata group, this is still an interesting study that provides novel insight into a potential mechanism determining initial evolutionary steps of gain and loss of the pyrenoid.

16S rRNA Gene Amplicon Sequencing of Gut Microbiota in Three Species of Deep-Sea Fish in Suruga Bay, Japan.

We report here 16S rRNA gene amplicon sequence analysis of the gut microbiota in three species of deep-sea fish collected from Suruga Bay, Japan. Of the three species, two were dominated by the phylum Proteobacteria (genus Photobacterium), while one was dominated by the phyla Spirochaetes (genus Brevinema) and Tenericutes (unclassified Mycoplasmataceae).

Transcriptome profile of carbon catabolite repression in an efficient l-(+)-lactic acid-producing bacterium Enterococcus mundtii QU25 grown in media with combinations of cellobiose, xylose, and glucose.

Enterococcus mundtii QU25, a non-dairy lactic acid bacterium of the phylum Firmicutes, is capable of simultaneously fermenting cellobiose and xylose, and is described as a promising strain for the industrial production of optically pure l-lactic acid (≥ 99.9%) via homo-fermentation of lignocellulosic hydrolysates. Generally, Firmicutes bacteria show preferential consumption of sugar (usually glucose), termed carbon catabolite repression (CCR), while hampering the catabolism of other sugars. In our previous study, QU25 exhibited apparent CCR in a glucose-xylose mixture phenotypically, and transcriptional repression of the xylose operon encoding initial xylose metabolism genes, likely occurred in a CcpA-dependent manner. QU25 did not exhibit CCR phenotypically in a cellobiose-xylose mixture. The aim of the current study is to elucidate the transcriptional change associated with the simultaneous utilization of cellobiose and xylose. To this end, we performed RNA-seq analysis in the exponential growth phase of E. mundtii QU25 cells grown in glucose, cellobiose, and/or xylose as either sole or co-carbon sources. Our transcriptomic data showed that the xylose operon was weakly repressed in cells grown in a cellobiose-xylose mixture compared with that in cells grown in a glucose-xylose mixture. Furthermore, the gene expression of talC, the sole gene encoding transaldolase, is expected to be repressed by CcpA-mediated CCR. QU25 metabolized xylose without using transaldolase, which is necessary for homolactic fermentation from pentoses using the pentose-phosphate pathway. Hence, the metabolism of xylose in the presence of cellobiose by QU25 may have been due to 1) sufficient amounts of proteins encoded by the xylose operon genes for xylose metabolism despite of the slight repression of the operon, and 2) bypassing of the pentose-phosphate pathway without the TalC activity. Accordingly, we have determined the targets of genetic modification in QU25 to metabolize cellobiose, xylose and glucose simultaneously for application of the lactic fermentation from lignocellulosic hydrolysates.

Bacillus subtilis Nucleoid-Associated Protein YlxR Is Involved in Bimodal Expression of the Fructoselysine Utilization Operon (frlBONMD-yurJ) Promoter.

Bacteria must survive harsh environmental fluctuations at times and have evolved several strategies. "Collective" behaviors have been identified due to recent progress in single-cell analysis. Since most bacteria exist as single cells, bacterial populations are often considered clonal. However, accumulated evidence suggests this is not the case. Gene expression and protein expression are often not homogeneous, resulting in phenotypic heterogeneity. In extreme cases, this leads to bistability, the existence of two stable states. In many cases, expression of key master regulators is bimodal via positive feedback loops causing bimodal expression of the target genes. We observed bimodal expression of metabolic genes for alternative carbon sources. Expression profiles of the frlBONMD-yurJ operon driven by the frlB promoter (PfrlB), which encodes degradation enzymes and a transporter for amino sugars including fructoselysine, were investigated using transcriptional lacZ and gfp, and translational fluorescence reporter mCherry fusions. Disruption effects of genes encoding CodY, FrlR, RNaseY, and nucleoid-associated protein YlxR, four known regulatory factors for PfrlB, were examined for expression of each fusion construct. Expression of PfrlB-gfp and PfrlB-mCherry, which were located at amyE and its original locus, respectively, was bimodal; and disruption of ylxR resulted in the disappearance of the clear bimodal expression pattern in flow cytometric analyses. This suggested a role for YlxR on the bimodal expression of PfrlB. The data indicated that YlxR acted on the bimodal expression of PfrlB through both transcription and translation. YlxR regulates many genes, including those related to translation, supporting the above notion. Depletion of RNaseY abolished heterogenous expression of transcriptional PfrlB-gfp but not bimodal expression of translational PfrlB-mCherry, suggesting the role of RNaseY in regulation of the operon through mRNA stability control and regulatory mechanism for PfrlB-mCherry at the translational level. Based on these results, we discuss the meaning and possible cause of bimodal PfrlB expression.