LuxT is a Global Regulator of Low-Cell Density Behaviors Including Type III Secretion, Siderophore Production, and Aerolysin Secretion in Vibrio harveyi

Quorum sensing (QS) is a chemical communication process in which bacteria produce, release, and detect extracellular signaling molecules called autoinducers. Via combined transcriptional and post-transcriptional regulatory mechanisms, QS allows bacteria to collectively alter gene expression on a population-wide scale. Recently, the LuxT transcription factor was shown to control V. harveyiqrr1, encoding the Qrr1 small RNA that functions at the core of the QS regulatory cascade. Here, we use RNA-Sequencing to reveal that, beyond control of qrr1, LuxT is a global regulator of 414 V. harveyi genes including those involved in type III secretion, siderophore production, and aerolysin toxin biosynthesis. Importantly, LuxT directly represses swrZ, encoding a transcription factor, and LuxT control of type III secretion, siderophore, and aerolysin genes occurs by two mechanisms, one that is SwrZ-dependent and one that is SwrZ-independent. All of these target genes specify QS-controlled behaviors that are enacted when V. harveyi is at low cell density. Thus, LuxT and SwrZ function in parallel with QS to drive particular low cell density behaviors. Phylogenetic analyses reveal that luxT is highly conserved among Vibrionaceae, but swrZ is less well conserved. In a test case to examine the relationship between LuxT and SwrZ, we find that in Aliivibrio fischeri, LuxT also functions as a swrZ repressor, and LuxT activates A. fischeri siderophore production via swrZ repression. Our results indicate that LuxT is a major regulator among Vibrionaceae, and, in the species that also possess swrZ, LuxT functions with SwrZ to control gene expression.

Auto/paracrine factors and early Wnt inhibition promote cardiomyocyte differentiation from human induced pluripotent stem cells at initial low cell density

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) have received increasing attention for their clinical use. Many protocols induce cardiomyocytes at an initial high cell density (confluence) to utilize cell density effects as hidden factors for cardiomyocyte differentiation. Previously, we established a protocol to induce hiPSC differentiation into cardiomyocytes using a defined culture medium and an initial low cell density (1% confluence) to minimize the hidden factors. Here, we investigated the key factors promoting cardiomyocyte differentiation at an initial low cell density to clarify the effects of cell density. Co-culture of hiPSCs at an initial low cell density with those at an initial high cell density showed that signals secreted from cells (auto/paracrine factors) and not cell–cell contact signals, played an important role in cardiomyocyte differentiation. Moreover, although cultures with initial low cell density showed higher expression of anti-cardiac mesoderm genes, earlier treatment with a Wnt production inhibitor efficiently suppressed the anti-cardiac mesoderm gene expression and promoted cardiomyocyte differentiation by up to 80% at an initial low cell density. These results suggest that the main effect of cell density on cardiomyocyte differentiation is inhibition of Wnt signaling at the early stage of induction, through auto/paracrine factors.

Keloid-like dermatofibroma

Dermatofibroma is a common benign skin tumor, mainly occurring in young to middle-aged females. It is frequently localized in the lower extremities. A typical dermatofibroma usually presents itself as a single firm papule or nodule, of variable color, bluish, brownish, or pinkish. Its clinical, dermoscopic, and histological features usually allow easy diagnosis [1]. However, it is possible to observe some variations of these typical features. Keloid-like dermatofibroma is one of these atypical presentations rarely reported in the literature [2]. A 40-year-old patient with no previous medical history presented to our dermatology department with a lumbosacral lesion evolving for several months. A physical examination revealed a firm, well-demarcated, asymptomatic erythematous nodule, 5 × 12 mm in size, localized in the lumbosacral area (Fig. 1). The patient denied any trauma preceding the onset of the lesion. There was no personal or familial history of keloidal scars. A dermoscopic examination revealed erythema, telangiectatic vessels, a shiny white streak, and a brownish-yellow pigmentation (Fig. 2). A biopsy was performed. A histological examination revealed an atrophic epidermis. The dermis contained a fibroblastic proliferation of low cell density haphazardly arranged, located on a fibromatous background (Figs. 3 and 4). Dermatofibroma with a keloidal presentation was the diagnosis.

Regulatory small RNA, Qrr2 is expressed independently of sigma factor-54 and can function as the sole Qrr sRNA to control quorum sensing in Vibrio parahaemolyticus

Bacterial cells alter gene expression in response to changes in population density in a process called quorum sensing (QS). In Vibrio harveyi , LuxO, a low cell density activator of sigma factor-54 (RpoN), is required for transcription of five non-coding regulatory sRNAs, Qrr1-Qrr5, which each repress translation of the master QS regulator LuxR. Vibrio parahaemolyticus , the leading cause of bacterial seafood-borne gastroenteritis, also contains five Qrr sRNAs that control OpaR (the LuxR homolog), controlling capsule polysaccharide (CPS), motility, and metabolism. We show that in a Δ luxO deletion mutant, opaR was de-repressed and CPS and biofilm were produced. However, in a Δ rpoN mutant, opaR was repressed, no CPS was produced, and less biofilm production was observed compared to wild type. To determine why opaR was repressed, expression analysis in Δ luxO showed all five qrr genes were repressed, while in Δ rpoN the qrr2 gene was significantly de-repressed. Reporter assays and mutant analysis showed Qrr2 sRNA can act alone to control OpaR. Bioinformatics analysis identified a sigma-70 (RpoD) -35 -10 promoter overlapping the canonical sigma-54 (RpoN) -24 -12 promoter in the qrr2 regulatory region. The qrr2 sigma-70 promoter element was also present in additional Vibrio species indicating it is widespread. Mutagenesis of the sigma-70 -10 promoter site in the Δ rpoN mutant background, resulted in repression of qrr2. Analysis of qrr quadruple deletion mutants, in which only a single qrr gene is present, showed that only Qrr2 sRNA can act independently to regulate opaR . Mutant and expression data also demonstrated that RpoN and the global regulator, Fis, act additively to repress qrr2 . Our data has uncovered a new mechanism of qrr expression and shows that Qrr2 sRNA is sufficient for OpaR regulation. Importance The quorum sensing non-coding sRNAs are present in all Vibrio species but vary in number and regulatory roles among species. In the Harveyi clade, all species contain five qrr genes, and in V. harveyi these are transcribed by sigma-54 and are additive in function. In the Cholerae clade, four qrr genes are present, and in V. cholerae the qrr genes are redundant in function. In V. parahaemolyticus , qrr2 is controlled by two overlapping promoters. In an rpoN mutant, qrr2 is transcribed from a sigma-70 promoter that is present in all V. parahaemolyticus strains and in other species of the Harveyi clade suggesting a conserved mechanism of regulation. Qrr2 sRNA can function as the sole Qrr sRNA to control OpaR.

A Simple, Cost-Effective, and Automation-Friendly Direct PCR Approach for Bacterial Community Analysis

Understanding bacterial interactions and assembly in complex microbial communities using 16S rRNA sequencing normally requires a large experimental load. However, the current DNA extraction methods, including cell disruption and genomic DNA purification, are normally biased, costly, time-consuming, labor-intensive, and not amenable to miniaturization by droplets or 1,536-well plates due to the significant DNA loss during the purification step for tiny-volume and low-cell-density samples.

Regulatory small RNA, Qrr2, is expressed independently of sigma factor-54 and functions autonomously in Vibrio parahaemolyticus to control quorum sensing

Bacterial cells alter gene expression in response to changes in population density in a process called quorum sensing (QS). In Vibrio harveyi, LuxO, a low cell density activator of sigma factor-54 (RpoN), is required for transcription of five non-coding regulatory sRNAs, Qrr1-Qrr5, which each repress translation of the master QS regulator LuxR. Vibrio parahaemolyticus, the leading cause of bacterial seafood-borne gastroenteritis, also contains five Qrr sRNAs that control OpaR (the LuxR homolog), required for capsule polysaccharide (CPS) and biofilm production, motility, and metabolism. We show that in a ΔluxO deletion mutant, opaR was de-repressed and CPS and biofilm were produced. However, in a ΔrpoN mutant, opaR was repressed, no CPS was produced, and less biofilm production was observed compared to wild type. To determine why opaR was repressed, expression analysis in ΔluxO showed all five qrr genes were repressed, while in ΔrpoN the qrr2 gene was significantly de-repressed. Reporter assays and mutant analysis showed Qrr2 sRNA can act autonomously to control OpaR. Bioinformatics analysis identified a sigma-70 (RpoD) -35 -10 promoter overlapping the canonical sigma-54 (RpoN) promoter in the qrr2 regulatory region. Mutagenesis of the sigma-70 -10 promoter site in the ΔrpoN mutant background, resulted in repression of qrr2. Analysis of qrr quadruple deletion mutants, in which only a single qrr gene is present, showed that only Qrr2 sRNA can act autonomously to regulate opaR. Mutant and expression data also demonstrated that RpoN and the global regulator Fis act additively to repress qrr2. Our data has uncovered a new mechanism of qrr expression and shows that Qrr2 sRNA is sufficient for OpaR regulation.

PHYTOPLANKTON COMPOSITION OF MARICULTURE AREA IN SUNGAI UDANG, PENANG, MALAYSIA, THE NORTHERN STRAIT OF MALACCA

A study was conducted in the marine finfish cage culture area in Sungai Udang, Penang, at the northern part of the Malacca Straits to examine the phytoplankton composition and abundance especially for potentially harmful phytoplankton. Monthly sampling were taken from March 2016 to January 2017 at nine sampling stations. Physio-chemical parameters of surface seawater such as pH, dissolved oxygen, temperature, salinity, total suspended solids, and nutrients (nitrate, nitrite, ammonium, phosphate and silicate) were also measured. A total of 54 phytoplankton taxa were recorded, with 37 genera belonged to diatoms, 15 of dinoflagellates, and 2 of cyanobacteria. The composition of phytoplankton was dominated by diatoms (>85%) at all sampling stations throughout the sampling period. The phytoplankton abundance ranged between 2.6×103 cells L-1 and 5.8×106 cells L-1. The potentially harmful toxic phytoplankton observed throughout the sampling period are dinoflagellates Alexandrium spp., Prorocentrum micans and Dinophysis caudata and diatoms, Pseudo-nitzchia spp but in low cell density. A total of six bloom-forming phytoplankton that can potentially trigger mass mortality of cultured fish such as Akashiwo sanguinea, Chaetoceros spp., Ceratium furca, Ceratium fusus, Margalefidinium spp. and Karlodinium spp. recorded at this area were relatively low in cell densities. Furthermore, no fish kill incident was reported in the area from blooms of phytoplankton during the period of study. Even though potentially harmful phytoplankton present were in low densities, they may pose significant risks to aquaculture activity if there is a sudden bloom. Hence, a monitoring program should be implemented to provide early warning of harmful algae blooms and safeguard the aquaculture industry in Sungai Udang, Penang.

Improvement of Opal Multiplex Immunofluorescence Workflow for Human Tissue Sections

The Opal multiplex technique is an established methodology for the detection of multiple biomarkers in one section. The protocol encompasses iterative single stainings and heating-mediated removal of the primary and secondary antibodies after each staining round, leaving untouched the Opal fluorophores which are deposited onto the antigen of interest. According to our experience, repetitive heating of skin sections often results in tissue damage, indicating an urgent need for milder alternatives to strip immunoglobulins. In this study, we demonstrate that considerable heating-related damage was found not only in skin but also in tissues of different origin, mostly characterized by low cell density. Importantly, the morphology remained fully intact when sections were repetitively exposed to β-mercaptoethanol-containing stripping buffer instead of multiple heating cycles. However, target epitopes appeared sensitive at a differential degree to multiple treatments with stripping buffer, as shown by loss in staining intensity, but in all cases, the staining intensity could be restored by increment of the primary antibody concentrations. Application of β-mercaptoethanol-containing stripping buffer instead of heating for antibody removal markedly improved the quality of the Opal multiplex technique, as a substantial higher number of differently colored cells could be visualized within a well-conserved morphological context:

Production of prodigiosin pigment by Serratia marcescens is negatively associated with cellular ATP levels during high-rate, low-cell-density growth

Serratia marcescens is a facultatively anaerobic bacterium and the most recognized producer of the hydrophobic pigment prodigiosin. Previous work has shown that prodigiosin both increases ATP production during population lag phase and approximately doubles the stationary-phase cell yield. Here, we employed both batch and chemostat culture methods to investigate prodigiosin’s role during high rate growth at low cell density as peak cellular ATP levels decline. Batch culture experiments utilizing artificial pigment induction showed an ATP reduction during low cell density growth. In addition, pigment induction during fixed growth rate chemostat culture revealed a negative correlation between cellular levels of prodigiosin and ATP (r = −0.95). Variable growth rate chemostat experiments showed an inverse relationship between ATP per cell and prodigiosin per cell during low-density growth but a direct relationship during high-density growth. Rate modeling of chemostat data quantified the pigment’s effect on cellular levels of ATP for both population growth phases. Finally, prodigiosin production in a heterologous bacterium led to ATP decline. These data with intact cells complement the established in vitro proton import function of prodigiosin pigment and may indicate an energy-spilling function during high rate, low cell density growth.