Comparison of Survival of Campylobacter jejuni in the Phyllosphere with That in the Rhizosphere of Spinach and Radish Plants

Campylobacter jejuni has been isolated previously from market produce and has caused gastroenteritis outbreaks linked to produce. We have tested the ability of this human pathogen to utilize organic compounds that are present in leaf and root exudates and to survive in the plant environment under various conditions. Carbon utilization profiles revealed that C. jejuni can utilize many organic acids and amino acids available on leaves and roots. Despite the presence of suitable substrates in the phyllosphere and the rhizosphere, C. jejuni was unable to grow on lettuce and spinach leaves and on spinach and radish roots of plants incubated at 33°C, a temperature that is conducive to its growth in vitro. However, C. jejuni was cultured from radish roots and from the spinach rhizosphere for at least 23 and 28 days, respectively, at 10°C. This enteric pathogen also persisted in the rhizosphere of spinach for prolonged periods of time at 16°C, a temperature at which many cool-season crops are grown. The decline rate constants of C. jejuni populations in the spinach and radish rhizosphere were 10- and 6-fold lower, respectively, than on healthy spinach leaves at 10°C. The enhanced survival of C. jejuni in soil and in the rhizosphere may be a significant factor in its contamination cycle in the environment and may be associated with the sporadic C. jejuni incidence and campylobacteriosis outbreaks linked to produce.     

Acyl Editing and Headgroup Exchange Are the Major Mechanisms That Direct Polyunsaturated Fatty Acid Flux into Triacylglycerols

Triacylglycerols (TAG) in seeds of Arabidopsis (Arabidopsis thaliana) and many plant species contain large amounts of polyunsaturated fatty acids (PUFA). These PUFA are synthesized on the membrane lipid phosphatidylcholine (PC). However, the exact mechanisms of how fatty acids enter PC and how they are removed from PC after being modified to participate in the TAG assembly are unclear, nor are the identities of the key enzymes/genes that control these fluxes known. By reverse genetics and metabolic labeling experiments, we demonstrate that two genes encoding the lysophosphatidylcholine acyltransferases LPCAT1 and LPCAT2 in Arabidopsis control the previously identified “acyl-editing” process, the main entry of fatty acids into PC. The lpcat1/lpcat2 mutant showed increased contents of very-long-chain fatty acids and decreased PUFA in TAG and the accumulation of small amounts of lysophosphatidylcholine in developing seeds revealed by [¹⁴C]acetate-labeling experiments. We also showed that mutations in LPCATs and the PC diacylglycerol cholinephosphotransferase in the reduced oleate desaturation1 (rod1)/lpcat1/lpcat2 mutant resulted in a drastic reduction of PUFA content in seed TAG, accumulating only one-third of the wild-type level. These results indicate that PC acyl editing and phosphocholine headgroup exchange between PC and diacylglycerols control the majority of acyl fluxes through PC to provide PUFA for TAG synthesis.Plant oils are an important natural resource to meet the increasing demands of food, feed, biofuel, and industrial applications (Lu et al., 2011; Snapp and Lu, 2012). The fatty acid composition in the triacylglycerols (TAG), especially the contents of polyunsaturated fatty acids (PUFA) or other specialized structures, such as hydroxy, epoxy, or conjugated groups, determines the properties and thus the uses of plant oils (Dyer and Mullen, 2008; Dyer et al., 2008; Pinzi et al., 2009; Riediger et al., 2009). To effectively modify seed oils tailored for different uses, it is necessary to understand the fundamental aspects of how plant fatty acids are synthesized and accumulated in seed oils.In developing oilseeds, fatty acids are synthesized in plastids and are exported into the cytosol mainly as oleic acid, 18:1 (carbon number:double bonds), and a small amount of palmitic acid (16:0) and stearic acid (18:0; Ohlrogge and Browse, 1995). Further modification of 18:1 occurs on the endoplasmic reticulum in two major pathways (Fig. 1): (1) the 18:1-CoA may be elongated into 20:1- to 22:1-CoA esters by a fatty acid elongase, FAE1 (Kunst et al., 1992); (2) the dominant flux of 18:1 in many oilseeds is to enter the membrane lipid phosphatidylcholine (PC; Shanklin and Cahoon, 1998; Bates and Browse, 2012), where they can be desaturated by the endoplasmic reticulum-localized fatty acid desaturases including the oleate desaturase, FAD2, and the linoleate desaturase, FAD3, to produce the polyunsaturated linoleic acid (18:2) and α-linolenic acid (18:3; Browse et al., 1993; Okuley et al., 1994). The PUFA may be removed from PC to enter the acyl-CoA pool, or PUFA-rich diacylglycerol (DAG) may be derived from PC by removal of the phosphocholine headgroup (Bates and Browse, 2012). The PUFA-rich TAG are then produced from de novo-synthesized DAG or PC-derived DAG (Bates and Browse, 2012) and PUFA-CoA by the acyl-CoA:diacylglycerol acyltransferases (DGAT; Hobbs et al., 1999; Zou et al., 1999). Alternatively, PUFA may be directly transferred from PC onto DAG to form TAG by an acyl-CoA-independent phospholipid:diacylglycerol acyltransferase (PDAT; Dahlqvist et al., 2000). Recent results demonstrated that DGAT and PDAT are responsible for the majority of TAG synthesized in Arabidopsis (Arabidopsis thaliana) seeds (Zhang et al., 2009).Open in a separate windowFigure 1.Reactions involved in the flux of fatty acids into TAG. De novo glycerolipid synthesis is shown in white arrows, acyl transfer reactions are indicated by dashed lines, and the movement of the lipid glycerol backbone through the pathway is shown in solid lines. Major reactions (in thick lines) controlling the flux of fatty acid from PC into TAG are as follows: LPC acylation reaction of acyl editing by LPCAT (A); PC deacylation reaction of acyl editing by the reverse action of LPCAT or phospholipase A (B); and the interconversion of DAG and PC by PDCT (C). Substrates are in boldface, enzymatic reactions are in italics. FAD, Fatty acid desaturase; FAS, fatty acid synthase; GPAT, acyl-CoA:G3P acyltransferase; LPA, lysophosphatidic acid; LPAT, acyl-CoA:LPA acyltransferase; PA, phosphatidic acid; PLC, phospholipase C; PLD, phospholipase D.The above TAG synthesis model highlights the importance of acyl fluxes through PC for PUFA enrichment in plant oils. However, the exact mechanisms of how fatty acids enter PC and how they are removed from PC after being modified to participate in the TAG assembly are unclear, nor are the identities of the enzymes/genes that control these fluxes known. The traditional view is that 18:1 enters PC through de novo glycerolipid synthesis (Fig. 1; Kennedy, 1961): the sequential acylation of glycerol-3-phosphate (G3P) at the sn-1 and sn-2 positions produces phosphatidic acid; subsequent removal of the phosphate group at the sn-3 position of phosphatidic acid by phosphatidic acid phosphatases (PAPs) produces de novo DAG; finally, PC is formed from DAG by a cytidine-5′-diphosphocholine:diacylglycerol cholinephosphotransferase (CPT; Slack et al., 1983; Goode and Dewey, 1999). However, metabolic labeling experiments in many different plant tissues by us and others (Williams et al., 2000; Bates et al., 2007, 2009; Bates and Browse, 2012; Tjellström et al., 2012) have demonstrated that the majority of newly synthesized fatty acids (e.g. 18:1) enter PC by a process termed “acyl editing” rather than by proceeding through de novo PC synthesis. Acyl editing is a deacylation-reacylation cycle of PC that exchanges the fatty acids on PC with fatty acids in the acyl-CoA pool (Fig. 1, A and B). Through acyl editing, newly synthesized 18:1 can be incorporated into PC for desaturation and PUFA can be released from PC to the acyl-CoA pool to be utilized for glycerolipid synthesis.Additionally, there is accumulating evidence that many plants utilize PC-derived DAG to synthesize TAG laden with PUFA (Bates and Browse, 2012). PC-derived DAG may be synthesized through the reverse reaction of the CPT (Slack et al., 1983, 1985) or by the phospholipases C and D (followed by PAP). However, our recent discovery indicates that the main PC-to-DAG conversion is catalyzed by a phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) through the phosphocholine headgroup exchange between PC and DAG (Fig. 1C; Lu et al., 2009; Hu et al., 2012). The PDCT is encoded by the REDUCED OLEATE DESATURATION1 (ROD1) gene (At3g15820) in Arabidopsis, which is responsible for about 40% of the flux of PUFA from PC through DAG into TAG synthesis (Lu et al., 2009). Acyl editing and PC-DAG interconversion through PDCT may work together to generate PUFA-rich TAG in oilseed plants (Bates and Browse, 2012).The enzymes/genes involved in the incorporation of 18:1 into PC through acyl editing are not known. However, stereochemical localization of newly synthesized fatty acid incorporation into PC predominantly at the sn-2 position (Bates et al., 2007, 2009; Tjellström et al., 2012) strongly suggest that the acyl editing cycle proceeds through the acylation of lysophosphatidylcholine (LPC) by acyl-CoA:lysophosphatidylcholine acyltransferases (LPCATs [Enzyme Commission 2.3.1.23]; Fig. 1A). High LPCAT activity has been detected in many different oilseed plants that accumulate large amounts of PUFA in TAG (Stymne and Stobart, 1987; Bates and Browse, 2012), suggesting the potential ubiquitous involvement of LPCAT in the generation of PUFA-rich TAG. Several possible pathways for the removal of acyl groups from PC to generate the lysophosphatidylcholine within the acyl editing cycle have been proposed. The acyl groups may be released from PC to enter the acyl-CoA pool via the reverse reactions of LPCATs (Stymne and Stobart, 1984) or by reactions of phospholipase A (Chen et al., 2011) followed by the acyl-CoA synthetases (Shockey et al., 2002). The main focus of this study was to identify the genes and enzymes involved in the incorporation of fatty acids into PC through acyl editing in Arabidopsis and to quantify the contribution of acyl editing and PDCT-based PC-DAG interconversion to controlling the flux of PUFA from PC into TAG. Herein, we demonstrate that mutants of two Arabidopsis genes encoding LPCATs (At1g12640 [LPCAT1] and At1g63050 [LPCAT2]) have reduced TAG PUFA content. Analysis of the acyl-editing cycle through metabolic labeling of developing seeds with [¹⁴C]acetate indicate that the lpcat1/lpcat2 double mutant was devoid of acyl editing-based incorporation of newly synthesized fatty acids into PC, indicating that these two genes are responsible for the acylation of LPC during acyl editing. Additionally, the triple mutant rod1/lpcat1/lpcat2 indicated that PDCT-based PC-DAG interconversion and acyl editing together provide two-thirds of the flux of PUFA from PC to TAG in Arabidopsis seeds.     

Speciation of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Multiple strains of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis isolated from animal, clinical, or food samples have been analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Whole bacterial cells were harvested from colonies or confluent growth on agar and transferred directly into solvent and then to a spot of dried 3-methoxy-4-hydroxycinnamic acid (matrix). Multiple ions in the 5,000- to 15,000-Da mass range were evident in spectra for each strain; one or two ions in the 9,500- to 11,000-Da range were consistently high intensity. “Species-identifying” biomarker ions (SIBIs) were evident from analyses of multiple reference strains for each of the six species, including the genome strains C. jejuni NCTC 11168 and C. jejuni RM1221. Strains grown on nine different combinations of media and atmospheres yielded SIBI masses within ±5 Da with external instrument calibration. The highest-intensity C. jejuni SIBIs were cytosolic proteins, including GroES, HU/HCj, and RplL. Multiple intraspecies SIBIs, corresponding probably to nonsynonymous nucleotide polymorphisms, also provided some intraspecies strain differentiation. MALDI-TOF MS analysis of 75 additional Campylobacter strains isolated from humans, poultry, swine, dogs, and cats revealed (i) associations of SIBI type with source, (ii) strains previously speciated incorrectly, and (iii) “strains” composed of more than one species. MALDI-TOF MS provides an accurate, sensitive, and rapid method for identification of multiple Campylobacter species relevant to public health and food safety.     

Chromosome numbers in callirhoe (Malvaceae)

Forty-four chromosome counts from the North American genusCallirhoe are reported, including first-recorded counts ofC. alcaeoides, C. digitata, C. bushii, C. triangulata, andC. involucrata var.tenuissima. Information concerning chromosome numbers from present and previous reports is summarized and evaluated. Observations are presented on meiosis for the genus in general, for male sterile individuals of gynodioecious species, and for selected F₁ hybrids. Pollen fertility data are recorded for the latter. Chromosomally three species groups are recognized: an exinvolucellate, diploid species group withn=14; an involucellate, tetraploid and octoploid species pair,n=28 and 56; and an involucellate, diploid and tetraploid species group withn=15 and 30. Intrageneric relationships and those within the tribe Malveae subtribe Malvinae are discussed.     

Respiratory resistance with histamine challenge by single-breath and forced oscillation methods

Relaxed expirations were obtained from five anesthetized dogs under control conditions and during various rates of intravenous infusion of histamine. All volume vs. time curves obtained from 20 ms to 2 s after the start of expiration were poorly described by a single exponential function but were fitted very well by a biexponential function. The resistance of the respiratory system as a function of frequency from 2 to 26 Hz was also determined by the forced oscillation method in the same dogs. Three two-compartment models of the respiratory system were identified from the exponentials fitted to the relaxed expiration data, and the one that had the most plausible parameter values under control conditions consisted of a homogeneous lung compartment connected to a viscoelastic compartment. Although a two-compartment model is arguably appropriate for describing relaxed expirations in normal dogs, physiological considerations suggest that there should be more than two interacting components with histamine infusion. We cannot identify all these components from our data, however. The equivalent complex impedance of the respiratory system was also calculated from the biexponential curves and showed significant variation in resistance over the frequency range from 0 to 2 Hz and negligible variation above 2 Hz. The calculated resistances at 2 Hz were consistently higher than those obtained by the forced oscillation method, which may be due to the nonlinear behavior of the respiratory system during relaxed expiration. We conclude that the single-breath and forced oscillation methods should be viewed as providing complimentary information about respiratory resistance.     

Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells

The response of malignant and nonmalignant human breast cell lines to the growth inhibitory effects of monoclonal antibodies against the epidermal growth factor (EGF) receptor was studied. A series of human breast cell lines, which express EGF receptor, were used: MDA-468, MDA-231, and Hs578T human breast cancer cells and the transformed human mammary epithelial cell lines 184A1N4 and 184A1N4-T that have been benzo[a]pyrene immortalized and further transformed with SV40T, respectively. Four antibodies of two different classes were tested: 225 immunoglobulin G (IgG), 108.4 IgG, 96 immunoglobulin M (IgM), and 42 IgM. All four antibodies inhibited the anchorage-dependent and -independent, EGF-stimulated growth of 184A1N4 and 184A1N4-T cells, respectively, and this growth inhibition could be reversed by the addition of increasing concentrations of EGF. In contrast, the antibodies inhibited the anchorage-dependent and -independent growth of MDA-468 cells in the absence of exogenous EGF suggesting that the antibodies were acting to block access of an endogenously produced ligand to the EGF receptor. In the presence of antibody and increasing concentrations of EGF, MDA-468 cell growth was first stimulated then inhibited as the EGF concentration increased, thus, uncovering the growth stimulatory potential of low concentrations of EGF in these cells. Data is presented that indicates MDA-468 cells secrete a transforming growth factor with autocrine growth stimulatory capabilities. The growth of MDA-231 and Hs578T cells, which contain activated ras oncogenes, was not inhibited by the antibodies and the growth of these cell lines was not stimulated by EGF. Of the cell lines studied only MDA-468 cells appear to possess an autocrine growth stimulatory capacity.     

The viable but nonculturable state of Kanagawa positive and negative strains of Vibrio parahaemolyticus

Ingestion of shellfish-associated Vibrio parahaemolyticus is the primary cause of potentially severe gastroenteritis in many countries. However, only Kanagawa phenomenon (hemolysin) positive (KP+) strains of V. parahaemolyticus are isolated from patients, whereas >99% of strains isolated from the environment do not produce this hemolysin (i.e. are KP-). The reasons for these differences are not known. Following a temperature downshift, Vibrio parahaemolyticus enters the viable but nonculturable (VBNC) state wherein cells maintain viability but cannot be cultured on routine microbiological media We speculated that KP+ and KP- strains may respond differently to the temperature and salinity conditions of seawater by entering into this state which might account for the low numbers of culturable KP+ strains isolated from estuarine waters. The response of eleven KP+ and KP- strains of V. parahaemolyticus following exposure to a nutrient and temperature downshift in different salinities, similar to conditions encountered in their environment, was examined. The strains included those from which the KP+ genes had been selectively removed or added. Our results indicated that the ability to produce hemolysin did not affect entrance into the VBNC state. Further, VBNC cells of both biotypes could be restored to the culturable state following an overnight temperature upshift.     

Endoproteolytic Processing of the Ebola Virus Envelope Glycoprotein: Cleavage Is Not Required for Function

Proteolytic processing is required for the activation of numerous viral glycoproteins. Here we show that the envelope glycoprotein from the Zaire strain of Ebola virus (Ebo-GP) is proteolytically processed into two subunits, GP₁ and GP₂, that are likely covalently associated through a disulfide linkage. Murine leukemia virions pseudotyped with Ebo-GP contain almost exclusively processed glycoprotein, indicating that this is the mature form of Ebo-GP. Mutational analysis identified a dibasic motif, reminiscent of furin-like protease processing sites, as the Ebo-GP cleavage site. However, analysis of Ebo-GP processing in LoVo cells that lack the proprotein convertase furin demonstrated that furin is not required for processing of Ebo-GP. In sharp contrast to other viral systems, we found that an uncleaved mutant of Ebo-GP was able to mediate infection of various cell lines as efficiently as the wild-type, proteolytically cleaved glycoprotein, indicating that cleavage is not required for the activation of Ebo-GP despite the conservation of a dibasic cleavage site in all filoviral envelope glycoproteins.     

Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota

Vertebrates harbor abundant lipopolysaccharide (LPS) in their gut microbiota. Alkaline phosphatases can dephosphorylate and detoxify the endotoxin component of LPS. Here, we show that expression of the zebrafish intestinal alkaline phosphatase (Iap), localized to the intestinal lumen brush border, is induced during establishment of the gut microbiota. Iap-deficient zebrafish are hypersensitive to LPS toxicity and exhibit the excessive intestinal neutrophil influx characteristic of wild-type zebrafish exposed to LPS. Both of these Iap mutant phenotypes are dependent on Myd88 and Tumor Necrosis Factor Receptor (Tnfr), proteins also involved in LPS sensitivity in mammals. When reared germ-free, the intestines of Iap-deficient zebrafish are devoid of neutrophils. Together, these findings demonstrate that the endogenous microbiota establish the normal homeostatic level of neutrophils in the zebrafish intestine through a process involving Iap, Myd88, and Tnfr. Thus, by preventing inflammatory responses, Iap plays a crucial role in promoting mucosal tolerance to resident gut bacteria.