Early Exponential Phase Research Articles

Short, auto-inducible promoters for well-controlled protein expression in Escherichia coli.

Expression of recombinant proteins in Escherichia coli often requires use of inducible promoters to shorten the lag phase and improve protein productivity and final protein titer. Synthetic molecules that cannot be metabolized by E. coli, such as isopropyl thiogalactopyranoside (IPTG), have been frequently used to trigger the protein expression during early exponential growth phase. This practice has many drawbacks, including high cost and toxicity of IPTG, complex operating procedure, and non-uniform protein expression pattern (some cells in the population do not express recombinant proteins). A few auto-inducible protein expression systems have been developed recently to overcome some of these limitations, but they required use of an additional plasmid or presence of large (a few kilobases) DNA part to be functional, making plasmid construction to be difficult, especially when multiple genes need to be expressed. In this study, by using RNA sequencing, we identified a short, endogenous promoter (PthrC) that can be auto-induced during early exponential growth phase, and improved its performance by use of native and mutated regulatory elements. We found that the developed mutants of PthrC drove uniform protein expression-close to 100% of cells were fluorescent when green fluorescence protein was used as target protein-and cells carrying them could achieve much higher cell density than those with T7 promoter (PT7), a commonly used inducible promoter. In terms of promoter strength (product protein quantity per cell), the developed promoter mutants can cover a range of strength, from 30 to 150% of maximal strength of PT7. One strong mutant (PthrC3_8) was found to work well at a large range of temperature (22, 30, 37°C) and in various media, and was also confirmed to cause less stress to host cell than PT7 when they were used to express a toxic protein. We foresee that PthrC3 and its mutants will be useful genetic parts for various applications including metabolic engineering and biocatalysis.

Production optimization of broad spectrum bacteriocin of three strains of Lactococcus lactis isolated from homemade buttermilk

Abstract Three potent strains of lactic acid bacteria (LAB) with bacteriocin producing abilities have been isolated from homemade buttermilk. They can produce proteinaceous bacteriocins in the medium which are resistant to heat (121 °C for 15 min) and can tolerate range of pH from 3.0 to 11.0. They were found to be Gram positive, non-motile, non-endospore forming, coccoid structures. The strains LABW1 and LABW3 were present in chains while LABW4 were present singly or in pairs. By considering the 16S rDNA sequence homologies and phylogenetic analysis LABW1 and LABW3 were identified as Lactococcus lactis subsp. hordniae where as LABW4 was identified as Lactococcus lactis subsp. lactis. Maximum extracellular bacteriocin production by the isolates takes place in MRS medium (pH 5.0–7.0) at 28 °C and it showed strong correlation with bacterial growth. They started producing bacteriocin at early exponential phase (400 AU/ml) and reached its peak at the early stationary phase (12800 AU/ml for LABW1 and LABW3 and 25600 AU/ml for LABW4) which remain unchanged up to late stationary phase. The bacteriocin of the isolates had an antibacterial spectrum both against Gram positive as well as Gram negative pathogenic bacterial strains. The inhibition of Gram negative bacteria by bacteriocin of LAB is an unusual phenomenon. It exhibits bacteriocidal action on Staphylococcus aureus MTCC96 and Pseudomonas aeruginosa MTCC741. Prominent morphological aberrations, cell shrinkages and pore formations on cell wall of pathogenic bacterial cells upon bacteriocin treatment as evidenced from scanning electron microscope (SEM).

Investigating Impact of Mycobacterial Physiology on Mycobacteriophage Life Cycles by Mass Spectrometry

Phage‐bacteria interaction plays an important role in both ecology and evolution. Phages lyse bacterial cells to keep nutrient and energy flows of the environment. Arm‐race between phages and bacteria drives evolution of both phages and bacteria. Current phage infection model demonstrates two typical types of phage‐bacteria interaction: lytic and lysogenic cycles. However, the model emphasizes the actions of individual phage particle and bacterial cell. Several researches have unveiled that phage‐bacteria interaction is behavior of both phage and bacterial communities, and a portion of bacterial cells can turn unsusceptible for phage infection. Since phage infection is initiated after the attachment of phage particles to bacterial cells, bacterial surface properties may impact phage‐bacteria interaction. Mycobacterium smegmatis (M. smegmatis) has a thick lipid‐rich cell envelop. Previous researches elucidated that glycopeptidolipids (GPLs) in cell envelope are involved in cell surface properties, receptors of phages and colony morphology of M. smegmatis. Therefore, GPLs may impact phage‐bacteria interaction. Besides GPLs, unique lipids including monomycoloyl trehaloses (MMTs) and triacyl glycerols (TAGs) of M. smegmatis may also impact phage‐bacteria interaction. As morphologies of both M. smegmatis cell and colony change with cell growth phase, composition of envelop lipids (GPLs, MMTs, TAGs etc.) and proteins may change with growth phage, which ultimately lead to alteration of phage‐bacteria interaction. To explore how the mycobacterial physiology affects mycobacteriophage life cycles, the impact of bacterial lipids and proteins composition change on phage and bacterial populations need to be investigated.Five mycobacteriophages from diverse clusters were selected to infect M. smegmatis cell cultures at early exponential and stationary phases for ten hours. Populations of both phages and bacterial cells were determined over time. After four and ten hours of infection, total proteins and lipids were extracted from subsamples of phage‐bacteria mixture, and further analyzed by mass spectrometry. The overall objective of this project is to figure out how phage life cycle alters when bacterial growth phase changes, and which proteins and lipids may be involved. The findings will provide a better understanding of phage‐host interactions over time and contribute towards novel applications in biotechnology.Support or Funding InformationHoward Hughes Medical Institute, Science Education Alliance; Polytechnic Institute, Purdue University; Department of Agricultural and Biological Engineering, Purdue University; Biotechnology Innovation and Regulatory Science Center, Purdue UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Repression of VvpM Protease Expression by Quorum Sensing and the cAMP-cAMP Receptor Protein Complex in Vibrio vulnificus.

Septicemia-causing Vibrio vulnificus produces at least three exoproteases, VvpE, VvpS, and VvpM, all of which participate in interactions with human cells. Expression of VvpE and VvpS is induced in the stationary phase by multiple transcription factors, including sigma factor S, SmcR, and the cAMP-cAMP receptor protein (cAMP-CRP) complex. Distinct roles of VvpM, such as induction of apoptosis, lead us to hypothesize VvpM expression is different from that of the other exoproteases. Its transcription, which was found to be independent of sigma S, is induced at the early exponential phase and then becomes negligible upon entry into the stationary phase. SmcR and CRP were studied regarding the control of vvpM expression. Transcription of vvpM was repressed by SmcR and cAMP-CRP complex individually, which specifically bound to the regions -2 to +20 and +6 to +27, respectively, relative to the vvpM transcription initiation site. Derepression of vvpM gene expression was 10- to 40-fold greater in an smcR crp double mutant than in single-gene mutants. Therefore, these results show that the expression of V. vulnificus exoproteases is differentially regulated, and in this way, distinct proteases can engage in specific interactions with a host.IMPORTANCE An opportunistic human pathogen, Vibrio vulnificus produces multiple extracellular proteases that are involved in diverse interactions with a host. The total exoproteolytic activity is detected mainly in the supernatants of the high-cell-density cultures. However, some proteolytic activity derived from a metalloprotease, VvpM, was present in the supernatants of the low-cell-density cultures sampled at the early growth period. In this study, we present the regulatory mechanism for VvpM expression via repression by at least two transcription factors. This type of transcriptional regulation is the exact opposite of those for expression of the other V. vulnificus exoproteases. Differential regulation of each exoprotease's production then facilitates the pathogen's participation in the distinct interactions with a host.

Thermal acclimation affects growth and lipophilic toxin production in a strain of cosmopolitan harmful alga Dinophysis acuminata

Species of the harmful algal bloom (HAB) genera Dinophysis are causative of one of the most widespread and expanding HAB events associated with the human intoxication, diarrheic shellfish poisoning (DSP). The effects of warming temperature on the physiology and toxinology of these mixotrophic species remain intractable due to their low biomass in nature and difficulties in establishing and maintaining them in culture. Hence, the present study investigated the influence of warming temperature, encompassing present and predicted climate scenarios, on growth and toxin production in a strain of the most cosmopolitan DSP-causative species, Dinophysis acuminata. The strain was isolated from western Japan, acclimated, and cultured over extended time spans.The specific growth and toxin production rates were highest at 20–26 °C and 17–29 °C, respectively, and had significant linear relationships during exponential phase. The cellular toxin production of okadaic acid and pectenotoxin-2 were highest during early exponential growth phase at temperatures ≤17 °C but highest during late stationary phase at temperatures ≥20 °C. The cellular toxin production of Dinophysistoxin-1, however, increased from early exponential to late stationary growth phase independently from temperature. The net toxin productions were not affected by acclimation temperature but significantly affected by growth and were highest during early exponential growth phase. Warming water temperatures increase growth and promote toxin production of D. acuminata, potentially increasing incidence of diarrheic shellfish poisoning events and closures of shellfish production. It is likely that D. acuminata is more toxic at low cell densities during bloom initiation in winter, and at high cell densities during bloom termination in spring-autumn. The results of the present research are also of importance for the mass production of D. acuminata for subsequent studies of the toxicological and pharmacological bioactivities of DSTs and PTX2, and the fate of these toxins in the natural environment and the vectoring shellfish molluscs.

Chromatin-level regulation of the fragmented dothistromin gene cluster in the forest pathogen Dothistroma septosporum.

Genes required for fungal secondary metabolite production are usually clustered, co-regulated and expressed in stationary growth phase. Chromatin modification has an important role in co-regulation of secondary metabolite genes. The virulence factor dothistromin, a relative of aflatoxin, provided a unique opportunity to study chromatin level regulation in a highly fragmented gene cluster that is switched on during early exponential growth phase. We analysed three histone modification marks by ChIP-qPCR and gene deletion in the pine pathogen Dothistroma septosporum to determine their effects on dothistromin gene expression across a time course and at different loci of the dispersed gene cluster. Changes in gene expression and dothistromin production were associated with changes in histone marks, with higher acetylation (H3K9ac) and lower methylation (H3K9me3, H3K27me3) during early exponential phase at the onset of dothistromin production. But while H3K27me3 directly influenced dothistromin genes dispersed across chromosome 12, effects of H3K9 acetylation and methylation were orchestrated mainly through a centrally located pathway regulator gene DsAflR. These results revealed that secondary metabolite production can be controlled at the chromatin-level despite the genes being dispersed. They also suggest that patterns of chromatin modification are important in adaptation of a virulence factor for a specific role in planta.

Prevention of Harmful Algal Blooms by Control of Growth Parameters

Harmful Algal Blooms (HAB) were investigated to prevent the outbreak of HAB in both freshwater and seawater. Parameters inducing HAB were volcanic eruption, sunlight, aeolian dust, environmental factors (current, pH, dissolved oxygen, food web, turbulence, growth phase), enzyme, iron, nutrients (carbon, nitrogen, phosphorus, sulfur, silicon, minerals) while the critical growth parameter for the outbreak of HAB was iron (Fe). HAB development was halted in freshwater and seawater due to the sulfur compounds (H2S, sulfates) inducing the deficiency of the dissolved Fe in the water. The atomic ratio of N/P is commonly known to be 16/1 in freshwater and 176/1 in seawater for HAB. Therefore, nitrogen can be a relatively limiting factor in seawater while phosphorus in freshwater. HAB could be prevented by control of growth parameters such as pH, temperature, sunlight, turbulence, nitrogen, phosphorus, iron, and sulfur compounds prior to reaching the early exponential phase of algal growth.

Indole at low concentration helps exponentially growing Escherichia coli survive at high temperature.

A culture of stationary phase Escherichia coli cells has been reported to produce copious indole when exposed to high temperature (50°C), and this response has been proposed to aid survival. We reinvestigated this phenomenon and found that indole production under these conditions is probably not a direct response to heat stress. Rather, E. coli produces indole when growth is prevented, irrespective of whether this is due to heat stress, antibiotic treatment or the removal of nutrients. Moreover, 300μM indole produced at 50°C does not improve the viability of heat stressed cells. Interestingly, a much lower concentration of indole (20 μM) improves the survival of an indole-negative strain (ΔtnaA) when heat stressed during exponential growth. In addition we have shown that the distribution of tryptophanase, the enzyme responsible for indole synthesis, is highly heterogeneous among cells in a population, except during the transition between exponential and stationary phases. The observation that, despite the presence of the tryptophanase, very little indole is produced during early exponential phase suggests that there is post-translational regulation of the enzyme.

INHIBITION OF BACTERIAL QUORUM SENSING BY THE RUMINAL FLUID OF CATTLE

Rumen fluid contains factors that inactivate autoinducers, monitor microflora development and prevent the mobilization of virulence properties, and thus, the formation of resistant bacteria. The aim of this study was to assess the ability of rumen fluid to suppress the intercellular communication of bacteria by inactivating the N-acyl-homoserine lactones. We used strains of luminescent Escherichia coli JLD271 which were transformed with plasmids containing quorum sensing genes and luminescence genes. Bacteria were cultivated in LB agar medium containing 10 ug/ml doxycycline then in LB medium until the early exponential growth phase. The autoinducers used were N-butyryl-L-homoserine lactone (C4-HSL), N-(3-oxo)-hexanoyl-L-homoserine lactone (oxo-С6-HSL) and N-dodecanoyl-L-homoserine lactone (C12-HSL). The ruminal fluid of young calves was collected via a fistula. Equal volumes of ruminal fluid and autoinducers were incubated then added to the cultures of luminescent bacterial strains. Dilution of the ruminal fluid up to 12.5% did not affect the luminescence of the bacterial biosensors. Incubation of ruminal fluid with N-acyl-homoserine lactones decreased their activity, marked by unexpressed specificity. The most effective inhibition of the N-(3-oxo)-hexanoyl-L-homoserine lactone was observed at 10-7 M, and above this concentration the efficiency of quorum sensing inhibition decreased. Ruminal fluid was able to inactivate Nacyl-homoserine lactones, and the efficiency of inhibition was not related to the length of the alkyl tail in the signal molecule. This suggests the ability of the ruminant digestive system to control bacteria by the inactivation of N-acyl-homoserine lactones, which regulate the quorum sensing system.

Multi-time series RNA-seq analysis of Enterobacter lignolyticus SCF1 during growth in lignin-amended medium.

The production of lignocellulosic-derived biofuels is a highly promising source of alternative energy, but it has been constrained by the lack of a microbial platform capable to efficiently degrade this recalcitrant material and cope with by-products that can be toxic to cells. Species that naturally grow in environments where carbon is mainly available as lignin are promising for finding new ways of removing the lignin that protects cellulose for improved conversion of lignin to fuel precursors. Enterobacter lignolyticus SCF1 is a facultative anaerobic Gammaproteobacteria isolated from tropical rain forest soil collected in El Yunque forest, Puerto Rico under anoxic growth conditions with lignin as sole carbon source. Whole transcriptome analysis of SCF1 during E.lignolyticus SCF1 lignin degradation was conducted on cells grown in the presence (0.1%, w/w) and the absence of lignin, where samples were taken at three different times during growth, beginning of exponential phase, mid-exponential phase and beginning of stationary phase. Lignin-amended cultures achieved twice the cell biomass as unamended cultures over three days, and in this time degraded 60% of lignin. Transcripts in early exponential phase reflected this accelerated growth. A complement of laccases, aryl-alcohol dehydrogenases, and peroxidases were most up-regulated in lignin amended conditions in mid-exponential and early stationary phases compared to unamended growth. The association of hydrogen production by way of the formate hydrogenlyase complex with lignin degradation suggests a possible value added to lignin degradation in the future.

An archaeal RNA binding protein, FAU-1, is a novel ribonuclease related to rRNA stability in Pyrococcus and Thermococcus

Ribosome biogenesis and turnover are processes necessary for cell viability and proliferation, and many kinds of proteins are known to regulate these processes. However, many questions still remain, especially in the Archaea. Generally, several ribonucleases are required to process precursor rRNAs to their mature forms, and to degrade rRNAs for quality control. Here, we found that FAU-1, which is known to be an RNA binding protein, possesses an RNase activity against precursor 5S rRNA derived from P. furiosus and T. kodakarensis in the order Thermococcales in vitro. An in vitro analysis revealed that UA sequences in the upstream of 5S rRNA were preferentially degraded by addition of FAU-1. Moreover, a fau-1 gene deletion mutant of T. kodakarensis showed a delay of exponential phase, reduction of maximum cell number and significant changes in the nucleotide sequence lengths of its 5S, 16S, and 23S rRNAs in early exponential phase. Our results suggest that FAU-1 is a potential RNase involved in rRNA stability through maturation and/or degradation processes.

FleQ regulates both the type VI secretion system and flagella in Pseudomonas putida.

Based on the analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry, flagellin FliC and a key member of the type VI secretion system Hcp1 were found to be secreted from Pseudomonas putida KT2442 cells in the late exponential phase, but in the early exponential phase only FliC was secreted. Relevant genes fleQ and rpoN were then individually deleted in P. putida KT2442, and the expression and transcription of fliC and hcp1 in these mutant strains were analyzed. In both △fleQ and △rpoN mutant cells, Hcp1 but not FliC was secreted in the late exponential phase, and it was secreted even in the early exponential phase. Transcriptomic analysis of △fleQ cells shows that most of the genes relevant to the type VI secretion system, including hcp1, were upregulated; several genes involved in 3',5'-cyclic diguanylic acid (c-di-GMP) synthesis and degradation were also significantly regulated. To further study the regulation process of the type VI secretion system, the phosphodiesterase BifA was overexpressed to lower the intracellular c-di-GMP level. The results demonstrate that FleQ positively regulates flagella biosynthesis but negatively regulates the type VI secretion system in P. putida. This finding is important for understanding the function and regulation of the type VI secretion system.

Functional and Catalytic Characterization of the Detoxifying Enzyme Haloalkane Dehalogenase from Rhizobium leguminosarum.

Haloalkane dehalogenases (EC 3.8.1.5, HLDs) are α/β-hydrolases which catalyze the irreversible cleavage of carbon-halogen bonds of haloalkanes, producing an alcohol, a halide and a hydrogen ion. Haloalkanes are acutely toxic to animals and humans and their toxic effects are mainly observed in the liver, kidneys and central nervous system. In the present work, the haloalkane dehalogenase from Rhizobium leguminosarum bv. trifolii (DrlA) was characterized. Reverse transcription polymerase chain reaction analysis and enzyme activity assays revealed that the DrlA gene expression in R. leguminosarum bv. trifolii is induced by 1,2- dibromoethane (1,2-DBE) during the early exponential phase. The gene of the enzyme was isolated, cloned and expressed in E. coli Rosetta (DE3). Recombinant DrlA displays its high catalytic activity towards 1,2-DBE and the long-chain haloalkane 1-iodohexane. Limited activity was observed for other aliphatic and cyclic haloalkanes, indicating that the enzyme displays restricted substrate specificity, compared to other bacterial HLDs. Homology modelling and phylogenetic analysis suggested that the enzyme belongs to the HLD-II subfamily and shares the same overall fold and domain organization as other bacterial HLDs, however major variations were identified at the hydrophobic substrate-binding cavity, the cap domain and the entrance of the main tunnel that affect the size of the active site pocket and the substrate recognition mechanism. This work sheds new light on the environmental fate and toxicity of 1,2-DBE and provides new knowledge on the structure, function and diversity of HLDs for developing applications in toxicology.

Fouling of extracellular algal organic matter during ultrafiltration: The influence of iron and the fouling mechanism

In this study, membrane fouling and the mechanism of algal extracellular organic matter (EOM) due to various trace heavy metals (iron) during ultrafiltration (UF) was investigated in detail. In both early and late exponential growth phases, the results indicated that the membrane fouling caused by EOM at low iron concentrations in this study was more severe than that at high and normal iron concentrations. Low iron concentrations produced the highest total (R) and reversible fouling resistance (Rre), of which Rre was higher, followed by membrane resistance (Rm), and irreversible fouling resistance (Rir). The analysis of EOM characteristics indicated that low iron in this study stimulated the growth of algae beyond high and normal iron concentrations, including increases in chlorophyll a; protein (tryptophan-like and tyrosine-like organic matter) content; and macro, medium and small molecular organic matters. Humic-like organics were more synthesized under high iron concentrations. Analyses of membrane fouling behavior illustrated that cake formation was the major fouling mechanism for the three iron concentrations, and it accounted for a greater proportion of fouling in the low iron concentration than in the other two iron concentrations; cake resistance played a more critical role in the late exponential growth phase than in the primary exponential phase.

Growth and adherence of Staphylococcus aureus were enhanced through the PGE2 produced by the activated COX-2/PGE2 pathway of infected oral epithelial cells.

Staphylococcus aureus is a major pathogen of varieties of oral mucous infection. Prostaglandin E2 (PGE2) is a pro-inflammatory factor and Cyclooxygenase 2 (COX-2) is a critical enzyme of PGE2 biosynthesis. The purpose of this study is to investigate whether Staphylococcus aureus can increase PGE2 production of oral epithelial cells and how PGE2 functions in the growth and adherence of Staphylococcus aureus. mRNA levels of COX-2, fnbpA and fnbpB were estimated by quantitative PCR. PGE2 production was measured by Enzyme Linked Immunosorbent Assay (ELISA). The binding biomass of Staphylococcus aureus to human fibronectin was investigated by crystal violet staining and confocal laser scanning microscopy and the adherent force was measured by atomic force microscope (AFM). The COX-2 mRNA level and PGE2 production were increased by Staphylococcus aureus. PGE2 promoted the growth and biofilm formation of Staphylococcus aureus, enhanced the attachment of Staphylococcus aureus to the human fibronectin as well as to the HOK cells. The transcription of fnbpB was up-regulated by PGE2 in both early and middle exponential phase but not fnbpA. These results suggest that the activation of COX-2/PGE2 pathway in oral epithelial cell by Staphylococcus aureus can in turn facilitate the growth and the ability to adhere of the pathogen. These findings uncover a new function of PGE2 and may lead to the potential of COX-2/PGE2 targeting in the therapy of inflammation and cancer in both which the COX-2/PGE2 pathway were observed activated.

Engineering of β-carotene hydroxylase and ketolase for astaxanthin overproduction in Saccharomyces cerevisiae

The conversion of β-carotene to astaxanthin is a complex pathway network, in which two steps of hydroxylation and two steps of ketolation are catalyzed by β-carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW) respectively. Here, astaxanthin biosynthesis pathway was constructed in Saccharomyces cerevisiae by introducing heterologous CrtZ and CrtW into an existing high β-carotene producing strain. Both genes crtZ and crtW were codon optimized and expressed under the control of constitutive promoters. Through combinatorial expression of CrtZ and CrtW from diverse species, nine strains in dark red were visually chosen from thirty combinations. In all the selected strains, strain SyBE_Sc118060 with CrtW from Brevundimonas vesicularis DC263 and CrtZ from Alcaligenes sp. strain PC-1 achieved the highest astaxanthin yield of 3.1 mg/g DCW. Protein phylogenetic analysis shows that the shorter evolutionary distance of CrtW is, the higher astaxanthin titer is. Further, when the promoter of crtZ in strain SyBE_Sc118060 was replaced from FBA1p to TEF1p, the astaxanthin yield was increased by 30.4% (from 3.4 to 4.5 mg/g DCW). In the meanwhile, 33.5-fold increase on crtZ transcription level and 39.1-fold enhancement on the transcriptional ratio of crtZ to crtW were observed at early exponential phase in medium with 4% (w/v) glucose. Otherwise, although the ratio of crtZ to crtW were increased at mid-, late-exponential phases in medium with 2% (w/v) glucose, the transcription level of both crtZ and crtW were actually decreased during the whole time course, consequently leading to no significant improvement on astaxanthin production. Finally, through high cell density fed-batch fermentation using a carbon source restriction strategy, the production of astaxanthin in a 5-L bioreactor reached to 81.0 mg/L, which was the highest astaxanthin titer reported in yeast. This study provides a reference to greatly enhance desired compounds accumulation by employing the key enzyme(s) in microbes. Open image in new window

Monitoring of Bioluminescent Lactobacillus plantarum in a Complex Food Matrix.

A bioluminescent Lactobacillus plantarum (pLuc2) strain was constructed. The luminescent signal started to increase during the early exponential phase and reached its maximum in the mid-exponential phase in a batch culture of the strain. The signal detection sensitivity of the strain was the highest in PBS (phosphate buffered saline), followed by milk and MRS broth, indicating that the sensitivity was influenced by the matrix effect. The strain was used in millet seed fermentation which has a complex matrix and native lactic acid bacteria (LAB). The luminescent signal was gradually increased until 9 h during fermentation and abolished at 24 h, indicating that the strain could be specifically tracked in the complex matrix and microflora. Therefore, the bioluminescent labeling system can be used for monitoring LAB in food and dairy sciences and industries.

Quantification of the d-Ala-d-Lac-Terminated Peptidoglycan Structure in Vancomycin-Resistant Enterococcus faecalis Using a Combined Solid-State Nuclear Magnetic Resonance and Mass Spectrometry Analysis.

Induction of vancomycin resistance in vancomycin-resistant enterococci (VRE) involves replacement of the d-Ala-d-Ala terminus of peptidoglycan (PG) stems with d-Ala-d-Lac, dramatically reducing the binding affinity of vancomycin for lipid II. Effects from vancomycin resistance induction in Enterococcus faecalis (ATCC 51299) were characterized using a combined solid-state nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) analysis. Solid-state NMR directly measured the total amounts of d-Lac and l,d-Ala metabolized from [2-13C]pyruvate, accumulated Park's nucleotide, and changes to the PG bridge-linking density during the early exponential growth phase (OD660 = 0.4) in intact whole cells of VRE. A high level of accumulation of depsipeptide-substituted Park's nucleotide consistent with the inhibition of the transglycosylation step of PG biosynthesis during the initial phase of vancomycin resistance was observed, while no changes to the PG bridge-linking density following the induction of vancomycin resistance were detected. This indicated that the attachment of the PG bridge to lipid II by the peptidyl transferases was not inhibited by the d-Ala-d-Lac-substituted PG stem structure in VRE. Compositions of mutanolysin-digested isolated cell walls of VRE grown with and without vancomycin resistance induction were determined by LC-MS. Muropeptides with PG stems terminating in d-Ala-d-Lac were found only in VRE grown in the presence of vancomycin. Percentages of muropeptides with a pentapeptide stem terminating in d-Ala-d-Lac for VRE grown in the presence of vancomycin were 26% for the midexponential phase (OD660 = 0.6) and 57% for the stationary growth phase (OD660 = 1.0). These high percentages indicate that d-Ala-d-Lac-substituted lipid II was efficiently utilized for PG biosynthesis in VRE.

Characterization of phosphatidic acid phosphatase activity in the oleaginous yeast Yarrowia lipolytica and its role in lipid biosynthesis.

Phosphatidic acid phosphatase (PAP) catalyses the committed step of triacylglycerol (TAG) biosynthesis and thus regulates the amounts of TAG produced by the cell. TAG is the target of biotechnological processes developed for the production of food lipids or biofuels. These processes are using oleaginous microorganisms like the yeast Yarrowia lipolytica as the TAG producers. Thus manipulating key enzymatic activities like PAP in Y. lipolytica could drive lipid biosynthesis towards TAG production and increase TAG yields. In this study, PAP activity in Y. lipolytica was characterized in detail and its role in lipid biosynthesis was addressed. PAP activity increased 2.5-fold with the addition of Mg2+ (1 mm) in the assay mixture, which means that most of the PAP activity was due to Mg2+ -dependent PAP enzymes (e.g. Pah1, App1). In contrast, N-ethylmaleimide (NEM) potently inhibited PAP activity, indicating the presence of NEM-sensitive PAP enzymes (e.g. App1, Lpp1). Localization studies revealed that the majority of PAP activity resides in the membrane fraction, while the cytosolic fraction harbours only a small amount of activity. PAP activity was regulated in a growth-dependent manner, being induced at the early exponential phase and declining thereafter. PAP activity did not correlate with TAG synthesis, which increased as cells progressed from the exponential phase to the early stationary phase. In stationary phase, TAG was mobilized with the concomitant synthesis of sterols and sterol esters. These results provide the first insights into the role of PAP in lipid biosynthesis by Y. lipolytica. Copyright © 2016 John Wiley & Sons, Ltd.

Growth Phase-dependent Variation of RNase BN/Z Affects Small RNAs: REGULATION OF 6S RNA

RNase BN, the RNase Z family member in E. coli, can participate in the processing of tRNA precursors. However, this function only becomes apparent when other processing enzymes are absent, raising the question of its primary physiological role. Here, we show that RNase BN itself is subject to growth phase-dependent regulation, because both rbn mRNA and RNase BN protein are at their highest levels in early exponential phase, but then decrease dramatically and are essentially absent in stationary phase. As a consequence of this variation, certain small RNAs, such as 6S RNA, remain low in exponential phase cells, and increase greatly in stationary phase. RNase BN affects 6S RNA abundance by decreasing its stability in exponential phase. RNase BN levels increase rapidly as cells exit stationary phase and are primarily responsible for the decrease in 6S RNA that accompanies this process. Purified RNase BN directly cleaves 6S RNA as shown by in vitro assays, and the 6S RNA:pRNA duplex is an even more favorable substrate of RNase BN. The exoribonuclease activity of RNase BN is unnecessary because all its action on 6S RNA is due to endonucleolytic cleavages. These data indicate that RNase BN plays an important role in determining levels of the global transcription regulator, 6S RNA, throughout the growth cycle.