Maintenance of Different Mannitol Uptake Systems during Starvation in Oxidative and Fermentative Marine Bacteria

The mannitol uptake systems in marine Vibrio and Pseudomonas isolates from the kelp beds off the South African west coast were examined. The fermentative Vibrio isolate possessed a constitutive rapid mannitol uptake system and also a soluble mannitol-1-phosphate dehydrogenase, indicative of a mannitol phosphotransferase system. An inducible, relatively less active mannitol uptake system was detected in the oxidative Pseudomonas isolate, and this strain possessed a mannitol dehydrogenase. The maintenance of these systems during starvation survival was studied. The Vibrio isolate maintained its initial uptake system for approximately 5 weeks of starvation, after which time the uptake system was replaced by one with a higher affinity for mannitol. The mannitol transport system of the Pseudomonas isolate was depressed early in starvation (30 h) but could be readily induced by exogenous mannitol after 6 weeks of starvation. The relative proportions of mannitol which was incorporated and respired were determined in starved Vibrio and Pseudomonas strains.     

Regulation of Sugar Transport Systems in Fusarium oxysporum var. lini

Fusarium oxysporum var. lini (ATCC 10960) formed a facilitated diffusion system for glucose (K_s, about 10 mM) when grown under repressed conditions. Under conditions of derepression, the same system was present together with a high-affinity (K_s, about 40 μM) active system. The maximum velocity of the latter was about 5% of that of the facilitated diffusion system. The high-affinity system was under the control of glucose repression and glucose inactivation. When lactose was the only carbon source in the medium, a facilitated diffusion system for lactose was found (K_s, about 30 mM).     

Self-organization at the origin of life

The concept of an (M,R) system with organizational invariance allows one to understand how a system may be able to maintain itself indefinitely if it is coupled to an external source of energy and materials. However, although this constitutes an important step towards understanding the difference between a living and a non-living system, it is not clear that an (M,R) system with organizational invariance is sufficient to define a living system. To take a further step towards defining what it means to be alive it is necessary to add to a simple (M,R) system some property that represents its identity, and which can be maintained and modified in subsequent generations.     

Curvularia Haloperoxidase: Antimicrobial Activity and Potential Application as a Surface Disinfectant

A presumed antimicrobial enzyme system, the Curvularia haloperoxidase system, was examined with the aim of evaluating its potential as a sanitizing agent. In the presence of hydrogen peroxide, Curvularia haloperoxidase facilitates the oxidation of halides, such as chloride, bromide, and iodide, to antimicrobial compounds. The Curvularia haloperoxidase system caused several-log-unit reductions in counts of bacteria (Pseudomonas spp., Escherichia coli, Serratia marcescens, Aeromonas salmonicida, Shewanella putrefaciens, Staphylococcus epidermidis, and Listeria monocytogenes), yeasts (Candida sp. and Rhodotorula sp.), and filamentous fungi (Aspergillus niger, Aspergillus tubigensis, Aspergillus versicolor, Fusarium oxysporum, Penicillium chrysogenum, and Penicillium paxilli) cultured in suspension. Also, bacteria adhering to the surfaces of contact lenses were killed. The numbers of S. marcescens and S. epidermidis cells adhering to contact lenses were reduced from 4.0 and 4.9 log CFU to 1.2 and 2.7 log CFU, respectively, after treatment with the Curvularia haloperoxidase system. The killing effect of the Curvularia haloperoxidase system was rapid, and 10⁶ CFU of E. coli cells/ml were eliminated within 10 min of treatment. Furthermore, the antimicrobial effect was short lived, causing no antibacterial effect against E. coli 10 min after the system was mixed. Bovine serum albumin (1%) and alginate (1%) inhibited the antimicrobial activity of the Curvularia haloperoxidase system, whereas glucose and Tween 20 did not affect its activity. In conclusion, the Curvularia haloperoxidase system is an effective sanitizing system and has the potential for a vast range of applications, for instance, for disinfection of contact lenses or medical devices.     

Control of gene expression in Helicobacter pylori using the Tet repressor

The lack of a versatile system to control gene expression in Helicobacter pylori has hampered efforts to study H. pylori physiology and pathogenesis. To overcome these limitations, we evaluated the utility of an inducible system based on the well-characterized Tet repressor (TetR) and Tet operator (tetO). As validation of this system, we introduced three copies of tetO into the promoter region upstream of the cagUT operon (encoding two virulence factors required for function of the H. pylori Cag type IV secretion system) and expressed tetR by introducing a codon-optimized gene into the chromosomal ureA locus. Introduction of the tetO copies upstream of cagUT did not disrupt promoter activity, as determined by immunoblotting for CagT. The subsequent introduction of tetR, however, did repress CagT synthesis. Production of CagT was restored when strains were cultured in the presence of the inducer, anhydrotetracycline. To demonstrate one potential application of this new tool, we analyzed the function of the Cag type IV secretion system. When the modified H. pylori strains were co-cultured with AGS cells, activity of the Cag type IV secretion system was dependent on the presence of anhydrotetracycline as evidenced by inducer-dependent induction of IL-8 secretion, CagA translocation, and appearance of type IV secretion system pili at the bacteria–host interface. These studies demonstrate the effectiveness of the tetR–tetO system to control gene expression in H. pylori and provide an improved system for studying H. pylori physiology and pathogenesis.     

Genetic studies on the loss of mu mutator activity in maize

Mutator activity of the Mu mutator system of maize can be lost by either outcrossing or inbreeding Mu stocks. The nature of these two kinds of Mu-loss phenomena was analyzed by testing the results of crossing Mu-loss stocks by active Mu lines. Outcross- Mu-loss stocks are capable of supporting Mu activity if crossed by an active mutator line. Inbred-Mu-loss stocks, however, inactivate the active Mu system contributed by a Mu line. Also, inbred- Mu-loss lines do not regain Mu activity after at least three generations of outcrossing to non-Mu stocks. These results suggest that, once the Mu system is inactivated by inbreeding, it remains inactivated for at least three generations of outcrossing. Further, once the system responsible for inactivation is established, it will, in turn, inactivate an active Mu system contributed by crossing with Mu plants. The outcross-Mu-loss does not seem to involve such an inactivation system. These results are interpreted in the light of recent evidence that Mu inactivation results from the modification of Mu 1 transposable elements involved in the Mu phenotype.     

In Vivo Programmed Gene Expression Based on Artificial Quorum Networks

The quorum sensing (QS) system, as a well-functioning population-dependent gene switch, has been widely applied in many gene circuits in synthetic biology. In our work, an efficient cell density-controlled expression system (QS) was established via engineering of the Vibrio fischeri luxI-luxR quorum sensing system. In order to achieve in vivo programmed gene expression, a synthetic binary regulation circuit (araQS) was constructed by assembling multiple genetic components, including the quorum quenching protein AiiA and the arabinose promoter P_araBAD, into the QS system. In vitro expression assays verified that the araQS system was initiated only in the absence of arabinose in the medium at a high cell density. In vivo expression assays confirmed that the araQS system presented an in vivo-triggered and cell density-dependent expression pattern. Furthermore, the araQS system was demonstrated to function well in different bacteria, indicating a wide range of bacterial hosts for use. To explore its potential applications in vivo, the araQS system was used to control the production of a heterologous protective antigen in an attenuated Edwardsiella tarda strain, which successfully evoked efficient immune protection in a fish model. This work suggested that the araQS system could program bacterial expression in vivo and might have potential uses, including, but not limited to, bacterial vector vaccines.     

Amino acid incorporation on 80s plant ribosomes: The complete system

A cell-free system directed by poly U or turnip yellow mosaic virus (TYMV)-RNA was obtained from imbibed seeds of Phaseolus aureus; this in vitro system was dependent upon exogenous tRNA. The poly U-directed system functioned in the presence of tRNAs from P. aureus, Vicia faba and yeast, whereas TYMV-RNA was translated only in the presence of tRNAs from P. aureus or V. faba. The pH and Mg²⁺ optima for aminoacylation of tRNAs of P. aureus, V. faba and yeast by leucine and phenylalanine were related to the overall pH and ionic concentration optima for the complete system.     

Inhibition of geranylgeranyl diphosphate synthesis in in vitro systems

The incorporation of [¹⁴C]mevalonate and [¹⁴C]isopentenyl diphosphate into geranylgeranyl diphosphate was investigated in in vitro systems from Cucurbita pepo (pumpkin) endosperm and from Avena sativa etioplasts. Mevalonate incorporation was effectively inhibited in the pumpkin system by geranylgeranyl diphosphate and geranylgeranyl monophosphate but less effectively by phytyl diphosphate or inorganic diphosphate. Membrane lipids, geranyllinalool, or lecithin enhanced mevalonate incorporation in the Cucurbita system. Incorporation of isopentenyl diphosphate was also enhanced by lecithin and inhibited by geranylgeranyl diphosphate in the Cucurbita system. No lipid enhancement was found in the Avena system; inhibition by GGPP required a much higher GGPP concentration than in the Cucurbita system.     

Distinct features of protein folding by the GroEL system from a psychrophilic bacterium, Colwellia psychrerythraea 34H

We investigated the protein folding mechanism of the GroEL system of a psychrophilic bacterium, Colwellia psychrerythraea 34H. The amount of mRNA of the groESL operon of C. psychrerythraea was increased about 6-fold after a temperature upshift from 8 to 18?°C for 30?min, suggesting that this temperature causes heat stress in this bacterium. A σ³²-type promoter was found upstream of the groESL, suggesting that the C. psychrerythraea groESL is regulated by the σ³² system, like the groESL in E. coli. The maximum ATPase and CTPase activities of CpGroEL were observed at 45 and 35?°C, respectively, which are much higher than the growth temperatures of C. psychrerythraea. We found that the refolding activity of the CpGroEL system in the presence of ATP is lower than that in the presence of CTP. This suggests that ATP is not the optimum energy source of the CpGroEL system. Analyses for the interaction of CpGroEL–CpGroES revealed that CTP could weaken this interaction, resulting in effective refolding function of the CpGroEL system. From these findings, we consider that the CpGroEL system possesses an energy-saving mechanism for avoiding excess consumption of ATP to ensure growth in a low-temperature environment.     

Fat metabolism in higher plants. Properties of the palmityl acyl carrier protein: stearyl acyl carrier protein elongation system in maturing safflower seed extracts

Palmityl acyl carrier protein is elongated specifically to stearyl acyl carrier protein by a system which required palmityl acyl carrier protein, malonyl CoA, and NADPH. Extracts from maturing safflower seeds, avocado mesocarp, and stroma from spinach chloroplasts contain the elongation system. The system differs from the de novo fatty acid synthetase system in that (1) it is inactivated at 37 °C whereas the de novo system remains fully active, (2) the pH optimum of the elongation system is 7.8–8.6 whereas the de novo system has a narrow pH optimum at 7.0, (3) NADPH is specifically required whereas the de novo system requires both NADPH and NADH, and (4) the elongation system is relatively insensitive to cerulenin whereas the de novo system is highly sensitive. Acetyl CoA does not serve as a C₂ donor. Stearyl acyl carrier protein, lauryl CoA, myristyl CoA, and palmityl CoA are inactive.     

Generation of Food-Grade Recombinant Lactic Acid Bacterium Strains by Site-Specific Recombination

The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (β-recombinase) and their respective target sites (attP-attB and six, respectively) is reported. The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented six sites, a multiple cloning site where passenger DNA could be inserted (e.g., the cI gene of bacteriophage A2), the int gene, and the attP site of phage A2. The clearing system provides a plasmid-borne gene encoding β-recombinase. The nonreplicative vector-borne delivery system was transformed into Lactobacillus casei ATCC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the attB site present in the genome of the host strain. The transfer of the clearing system into this strain, with the subsequent expression of the β-recombinase, led to site-specific DNA resolution of the non-food-grade DNA. These methods were validated by the construction of a stable food-grade L. casei ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation.     

Cloning and characterization of the BglII restriction–modification system reveals a possible evolutionary footprint

BglII, a type II restriction–modification (R–M) system from Bacillus globigii, recognizes the sequence 5′-AGATCT-3′. The system has been cloned into E. coli in multiple steps: first the methyltransferase (MTase) gene, bglIIM, was cloned from B. globigii RUB561, a variant containing an inactivated endonuclease (ENase) gene (bglIIR). Next the ENase protein (R.BglII) was purified to homogeneity from RUB562, a strain expressing the complete R–M system. Oligonucleotide probes specific for the 5′ end of the gene were then synthesized and used to locate bglIIR, and the gene was isolated and cloned in a subsequent step. The nucleotide sequence of the system has been determined, and several interesting features have been found. The genes are tandemly arranged, with bglIIR preceding bglIIM. The amino acid sequence of M.BglII is compared to those of other known MTases. A third gene encoding a protein with sequence similarity to known C elements of other R–M systems is found upstream of bglIIR. This is the first instance of a C gene being associated with an R–M system where the R and M genes are collinear. In addition, open reading frames (ORFs) resembling genes involved with DNA mobility are found in close association with BglII. These may shed light on the evolution of the R–M system.     

A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus

The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms.     

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.     

An Active Type I-E CRISPR-Cas System Identified in Streptomyces avermitilis

CRISPR-Cas systems, the small RNA-dependent immune systems, are widely distributed in prokaryotes. However, only a small proportion of CRISPR-Cas systems have been identified to be active in bacteria. In this work, a naturally active type I-E CRISPR-Cas system was found in Streptomyces avermitilis. The system shares many common genetic features with the type I-E system of Escherichia coli, and meanwhile shows unique characteristics. It not only degrades plasmid DNA with target protospacers, but also acquires new spacers from the target plasmid DNA. The naive features of spacer acquisition in the type I-E system of S. avermitilis were investigated and a completely conserved PAM 5’-AAG-3’ was identified. Spacer acquisition displayed differential strand bias upstream and downstream of the priming spacer, and irregular integrations of new spacers were observed. In addition, introduction of this system into host conferred phage resistance to some extent. This study will give new insights into adaptation mechanism of the type I-E systems in vivo, and meanwhile provide theoretical foundation for applying this system on the genetic modification of S. avermitilis.     

Evidence for horizontal transfer of SsuDAT1I restriction-modification genes to the Streptococcus suis genome

Different strains of Streptococcus suis serotypes 1 and 2 isolated from pigs either contained a restriction-modification (R-M) system or lacked it. The R-M system was an isoschizomer of Streptococcus pneumoniae DpnII, which recognizes nucleotide sequence 5′-GATC-3′. The nucleotide sequencing of the genes encoding the R-M system in S. suis DAT1, designated SsuDAT1I, showed that the SsuDAT1I gene region contained two methyltransferase genes, designated ssuMA and ssuMB, as does the DpnII system. The deduced amino acid sequences of M.SsuMA and M.SsuMB showed 70 and 90% identity to M.DpnII and M.DpnA, respectively. However, the SsuDAT1I system contained two isoschizomeric restriction endonuclease genes, designated ssuRA and ssuRB. The deduced amino acid sequence of R.SsuRA was 49% identical to that of R.DpnII, and R.SsuRB was 72% identical to R.LlaDCHI of Lactococcus lactis subsp. cremoris DCH-4. The four SsuDAT1I genes overlapped and were bounded by purine biosynthetic gene clusters in the following gene order: purF-purM-purN-purH-ssuMA-ssuMB-ssuRA-ssuRB-purD-purE. The G+C content of the SsuDAT1I gene region (34.1%) was lower than that of the pur region (48.9%), suggesting horizontal transfer of the SsuDAT1I system. No transposable element or long-repeat sequence was found in the flanking regions. The SsuDAT1I genes were functional by themselves, as they were individually expressed in Escherichia coli. Comparison of the sequences between strains with and without the R-M system showed that only the region from 53 bp upstream of ssuMA to 5 bp downstream of ssuRB was inserted in the intergenic sequence between purH and purD and that the insertion target site was not the recognition site of SsuDAT1I. No notable substitutions or insertions could be found, and the structures were conserved among all the strains. These results suggest that the SsuDAT1I system could have been integrated into the S. suis chromosome by an illegitimate recombination mechanism.     

Biphasic kinetic plots and specific analogs distinguishing and describing amino acid transport sites in S37 ascites tumor cells

Curve-fitting procedures indicated that exo-2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) modified V and K_m for one of two systems serving for histidine transport into the S37 ascites tumor cells. When this system was obliterated by leucine in the medium, BCH had no effect on histidine transport.Curve-fitting procedures similarly suggest N-methyl-α-aminoisobutyric acid affected the K_m and V values for the other histidine-transporting system and that carboxymethylhistidine (His(Cm)) inhibited both transport systems. His(Cm) further inhibited histidine uptake into leucine-inhibited cells. K_m and V values were altered simultaneously in the presence of several inhibitory analogs.Alanine methyl ester markedly inhibited high-concentration histidine uptake, whereas leucine methyl ester markedly inhibited low-concentration histidine uptake.The present results confirm earlier suggestions that our high c system is Christensen's A system and our low c system his L system. We also confirm a very high degree of specificity of N-methyl-α-aminoisobutyric acid for the A or high c system, and of BCH for the L or low c system. We suggest the utility of combining two approaches to the study of transport system properties; use of specific analogs and modification of biphasic plots. We demonstrate that the carboxyl group is not a prerequisite molecular feature for inhibitory interaction with the A or L system.     

Preparation of a Cell-Free, Isobutene-Forming System from Rhodotorula minuta

The cell-free isobutene-forming system of Rhodotorula minuta IFO 1102 was prepared. This system consisted of isovaleric acid, NADPH, and a cell extract. The maximal activity obtained by using the described cell-free system was 17 nl/mg of protein per h. It appears that this cell-free isobutene-forming system operates in living cells of R. minuta.