AI-2-dependent gene regulation in <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis>

Background  

Autoinducer 2 (AI-2), a widespread by-product of the LuxS-catalyzed S-ribosylhomocysteine cleavage reaction in the activated methyl cycle, has been suggested to serve as an intra- and interspecies signaling molecule, but in many bacteria AI-2 control of gene expression is not completely understood. Particularly, we have a lack of knowledge about AI-2 signaling in the important human pathogens Staphylococcus aureus and S. epidermidis.     

Autoantigen-specific memory CD4+ T cells are prevalent early in progression to Type 1 diabetes

Autoreactive CD4(+) T cells contribute to the destruction of insulin producing beta cells in Type 1 diabetes (T1D). Using MHC class II tetramers, we have analyzed the frequency of GAD65- (274-286; 555-567) and insulin- (A1-15; A6-21) specific CD4(+) T cells in 31 children with T1D, 65 multiple autoantibody-positive children and 93 HLA- and age-matched controls. In a smaller group of children T-cell responses of memory origin to the same autoantigens were investigated. We observed a higher response to GAD65 555-567 in the autoantibody-positive children than in the controls (P=0.017). Memory T-cell responses to GAD65 555-567 were more frequent among T1D patients (P=0.025) and autoantibody-positive (P=0.054), while all controls were negative (n=28). In summary, the presence of antigen experienced GAD65-specific T cells in the subjects with diabetes-associated autoimmunity is encouraging for further directions in the prediction of T1D.     

A systematic characterization of mitochondrial proteome from human T leukemia cells

Global understanding of tissue-specific differences in mitochondrial signal transduction requires comprehensive mitochondrial protein identification from multiple cell and tissue types. Here, we explore the feasibility and efficiency of protein identification using the one-dimensional gel electrophoresis in combination with the nano liquid-chromatography tandem mass spectrometry (GeLC-MS/MS). The use of only 40 mug of purified mitochondrial proteins and data analysis using stringent scoring criteria and the molecular mass validation of the gel slices enables the identification of 227 known mitochondrial proteins (membrane and soluble) and 453 additional proteins likely to be associated with mitochondria. Replicate analyses of 60 mug of mitochondrial proteins on the faster scanning LTQ mass spectrometer validate all the previously identified proteins and most of the single hit proteins except the 81 single hit proteins. Among the identified proteins, 466 proteins are known to functionally participate in various processes such as respiration, tricarboxylic acid cycle (TCA cycle), amino acid and nucleotide metabolism, glycolysis, protection against oxidative stress, mitochondrial assembly, molecular transport, protein biosynthesis, cell cycle control, and many known cellular processes. The distribution of identified proteins in terms of size, pI, and hydrophobicity reveal that the present analytical strategy is largely unbiased and very efficient. Thus, we conclude that this approach is suitable for characterizing subcellular proteomes form multiple cells and tissues.     

Increased C-telopeptide Cross-linking of Tendon Type I Collagen in Fibromodulin-deficient Mice

The controlled assembly of collagen monomers into fibrils, with accompanying intermolecular cross-linking by lysyl oxidase-mediated bonds, is vital to the structural and mechanical integrity of connective tissues. This process is influenced by collagen-associated proteins, including small leucine-rich proteins (SLRPs), but the regulatory mechanisms are not well understood. Deficiency in fibromodulin, an SLRP, causes abnormal collagen fibril ultrastructure and decreased mechanical strength in mouse tendons. In this study, fibromodulin deficiency rendered tendon collagen more resistant to nonproteolytic extraction. The collagen had an increased and altered cross-linking pattern at an early stage of fibril formation. Collagen extracts contained a higher proportion of stably cross-linked α1(I) chains as a result of their C-telopeptide lysines being more completely oxidized to aldehydes. The findings suggest that fibromodulin selectively affects the extent and pattern of lysyl oxidase-mediated collagen cross-linking by sterically hindering access of the enzyme to telopeptides, presumably through binding to the collagen. Such activity implies a broader role for SLRP family members in regulating collagen cross-linking placement and quantity.     

Mapping of Functional Domains in Herpesvirus Saimiri Complement Control Protein Homolog: Complement Control Protein Domain 2 Is the Smallest Structural Unit Displaying Cofactor and Decay-Accelerating Activities

Herpesvirus saimiri encodes a functional homolog of human regulator-of-complement-activation proteins named CCPH that inactivates complement by accelerating the decay of C3 convertases and by serving as a cofactor in factor I-mediated inactivation of their subunits C3b and C4b. Here, we map the functional domains of CCPH. We demonstrate that short consensus repeat 2 (SCR2) is the minimum domain essential for classical/lectin pathway C3 convertase decay-accelerating activity as well as for factor I cofactor activity for C3b and C4b. Thus, CCPH is the first example wherein a single SCR domain has been shown to display complement regulatory functions.The complement system is an ancient and yet highly evolved effector mechanism of immune defense that forms an imperative branch of innate immunity (23, 46). In addition, recent findings have clearly revealed its role as a vital viaduct between the innate and acquired immune systems (6, 18). Thus, it is not surprising that the system helps in purging a wide array of invaders, including viruses. Consequently, for their successful survival, many viruses have developed mechanisms to subvert the host complement system (7, 24, 26, 29, 39, 45). Herpesviruses and poxviruses, in particular, subvert host complement by encoding structural and/or functional homologs of human complement regulators belonging to the regulator-of-complement-activation (RCA) family, by capturing host membrane complement regulators and by using cellular receptors for entering cells (1, 8, 15, 23).The RCA proteins are formed by multiple tandem repeats of bead-like complement control protein (CCP) domains or short consensus repeats (SCRs) separated by short linkers. It has been suggested that the sequence variations enforced upon these SCR domain folds and the interdomain dynamics dictate the functionality of the complement regulators (17, 19, 44, 49). Because sequence similarity in herpesviral complement regulators varies between 43% and 89% and in poxviral complement regulators exceeds 91%, it is likely that the structural diversity in herpesviral complement regulators may have resulted in functional differences in these proteins and/or have resulted in variation in structural requirements for complement regulation. In the herpesviridae family, detailed functional characterization has been performed for complement regulators of Kaposi''s sarcoma-associated herpesvirus (Kaposica/KCP) (28, 42), herpesvirus saimiri (HVS) (CCPH) (10, 38), and rhesus rhadinovirus (RCP) (31). All these proteins showed conservation of complement regulatory activities, indicating thereby that structural diversity has not resulted in loss of complement regulatory functions in these proteins. However, it is not clear whether sequence variations within the herpesviral complement regulators have resulted in differences in the domain requirements for complement regulatory activities, since mapping of functional domains has been performed only for Kaposica (30, 43). In the present study, we therefore have mapped the complement regulatory domains of HVS CCPH to get further insight into diversity in domain requirements for functional activities.HVS is a classical prototype of the gamma 2-herpesviruses or rhadinoviruses. It causes rapidly progressing fulminant lymphoma, lymphosarcoma, and leukemia of T-cell origin in marmosets, owl monkeys, and other species of New World primates but not in its natural host, the squirrel monkey (9, 16). Unlike other herpesviruses, it encodes two complement regulators: an RCA homolog (ORF 4; CCPH) that regulates the early steps of complement activation (2, 10) and a CD59 homolog (ORF 15) that inhibits the late steps of complement activation (4, 36). The RCA homolog is formed of four SCR modules (Fig. ​(Fig.1).1). As a result of alternative splicing, the protein is expressed as a full-length membrane-bound form (mCCPH) containing the transmembrane region as well as a spliced secretory form (sCCPH) lacking the transmembrane region (2). Earlier, we showed that sCCPH inhibits complement by targeting C3 convertases: (i) it supports serine protease factor I-mediated inactivation of C3b and C4b, the subunits of C3 convertases (cofactor activity), and (ii) it accelerates the irreversible decay of the classical pathway (CP)/lectin pathway and to a limited extent the alternative pathway (AP) C3 convertases (decay-accelerating activity [DAA]) (38).Open in a separate windowFIG. 1.Schematic illustration of sCCPH and SDS-PAGE analysis of purified recombinant sCCPH and its deletion mutants. (Top) Schematic representation of the structure of the soluble form of CCPH (sCCPH), which is composed of four SCRs. The domains are numbered, and the minimum domains shown to be important for C3b and C4b cofactor activities (CFA) and CP DAA are identified. (Bottom) Expressed and purified sCCPH and its deletion mutants were analyzed by 12% (left) and 13% (right) SDS-PAGE under reducing conditions and stained with Coomassie blue. Molecular weights as determined by SDS-PAGE: for sCCPH, 32,000; for SCR1-3, 26,000; for SCR2-4, 27,500; for SCR1-2, 17,000; for SCR2-3, 17,500; for SCR3-4, 16,500; for SCR1, 9,500; for SCR2, 7,000; for SCR3, 8,000; and for SCR4, 8,000. Molecular mass is expressed as kilodaltons in the figure.(This work was done in partial fulfillment of the Ph.D. thesis requirements of A.K.S., University of Pune, Pune, India.)In order to map the functional domains of sCCPH, we have generated a series of soluble triple, double, and single SCR deletion mutants. In brief, the deletion mutants of sCCPH comprising SCR1-3, -2-4, -1-2, -2-3, and -3-4 as well as SCR1, -2, -3, and -4 were constructed from the full-length HVS sCCPH clone (38) by PCR amplification and cloning into the bacterial expression vector pET29. The authenticity of each of the clones was confirmed by DNA sequencing, and then they were transformed into the Escherichia coli BL21 strain for expression. The mutants carried the histidine tag at the C terminus and hence were purified to homogeneity by using histidine affinity chromatography. Refolding of the purified proteins was performed by using the rapid dilution method as previously described (38, 47, 48), and the refolded proteins were loaded onto a Superose 12 gel filtration column (Pharmacia) to obtain monodisperse populations of the expressed mutants (38, 48). The preservation of various functions in mutants (see below) suggests that the mutants have maintained their proper conformation. The expressed proteins were >95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Fig. ​(Fig.11).To identify the domains required for cofactor activities of sCCPH against C3b and C4b, we utilized a fluid phase assay wherein C3b or C4b was incubated with each of the deletion mutants and factor I, and inactivation of C3b/C4b (cleavage of the α′-chain) was determined by running the samples on SDS-PAGE gels. It is clear from the data presented in Fig. ​Fig.22 that sCCPH and the mutants SCR1-3, -2-4, and -1-2 supported the cleavage of the α′-chain of C3b. A very weak cleavage was also supported by SCR2-3 and -3-4. The cleavage of the α′-chain of C4b, however, was supported by sCCPH and the mutants SCR1-3, -2-4, -1-2, and -2-3 but not by SCR3-4 (Fig. ​(Fig.2).2). Together, these data point out that SCR1 and -2 considerably contribute to the C3b and C4b cofactor activities of sCCPH but that SCR3 and SCR4 in the case of C3b cofactor activity and SCR3 in the case of C4b cofactor activity contribute to its optimal activity. These results, however, did not elucidate whether a single domain(s) could impart the cofactor activities. We therefore expressed the single-domain mutants (SCR1, SCR2, SCR3, and SCR4) and analyzed their cofactor activities. The results presented in Fig. ​Fig.33 indicate that SCR2, by itself, possesses the ability to support factor I-mediated inactivation of C3b and C4b; SCR3 also displayed very weak cofactor activity against C3b when used at higher concentrations (88 μM; data not shown). These results suggest that structural elements involved in the interaction of sCCPH with factor I are primarily located within SCR2 and -3. Admittedly, the single-domain mutants possess very weak cofactor activities and other domains too contribute to the optimal activity; the cofactor activities of SCR2 for C3b and C4b were 781- and 212-fold lower than that for sCCPH (Fig. ​(Fig.3).3). It should be mentioned here that earlier observations on mapping of the human RCA proteins (factor H, C4b-binding protein, membrane cofactor protein, and complement receptor 1) (3, 11-13, 21), Kaposica (30), and vaccinia virus CCP (VCP) (27) indicated that a minimum of two (in Kaposica) or three (in all other RCA proteins) successive SCR domains are necessary for factor I cofactor activities. Thus, sCCPH is the first complement regulator in which a single SCR domain has been shown to display the factor I cofactor function.Open in a separate windowFIG. 2.Analysis of factor I cofactor activity of sCCPH and its deletion mutants for human complement proteins C3b and C4b. Cofactor activity was assessed by incubating 3.0 μg of human C3b (upper panels) or C4b (lower panels) with sCCPH/SCR1-3/SCR2-4 (4.0 μM) or SCR1-2/2-3/3-4 (24 μM) in the presence or absence of factor I (100 ng) for the indicated time periods at 37°C in 10 mM sodium phosphate, pH 7.4, containing 145 mM NaCl. The reactions were stopped by addition of sample buffer containing dithiothreitol, and the amount of C3b or C4b cleaved was visualized by subjecting the samples to SDS-PAGE analysis on 10% or 11.5% gel, respectively, and staining with Coomassie blue. During C3b cleavage, the α′-chain is cleaved into N-terminal 68-kDa and C-terminal 46-kDa fragments. The 46-kDa fragment is then cleaved into a 43-kDa fragment. These cleavages indicate inactivation of C3b. In the case of C4b, the α′-chain is cleaved into N-terminal 27-kDa, C-terminal 16-kDa (not visible in the gel), and central C4d fragments. These cleavages indicate the inactivation of C4b.Open in a separate windowFIG. 3.Analysis of factor I cofactor activity (CFA) of single SCR mutants of sCCPH for human complement proteins C3b and C4b. (Upper panels) Cofactor activity was assessed by incubating 3.0 μg of human C3b or C4b with the single SCR mutants (44 μM) in the presence or absence of factor I (100 ng) for 4 h at 37°C in PBS (10 mM sodium phosphate, pH 7.4, containing 145 mM NaCl). The reactions were stopped by addition of sample buffer containing dithiothreitol, and the amount of C3b or C4b cleaved was visualized by subjecting the samples to 13% SDS-PAGE and stained with Coomassie blue. Cleavage of the α′-chain of C3b and C4b and generation of cleavage products indicate the inactivation of these proteins. (Middle panels) Human C3b (3.0 μg) or C4b (3.0 μg) and factor I (100 ng) were incubated in PBS with increasing concentrations of sCCPH or the SCR2 mutant at 37°C for 1 h, and the cleavage products were analyzed as described above. (Lower panels) The intensity of the α′-chains of C3b and C4b in the middle panels was determined densitometrically and is represented graphically. The closed and open circles represent sCCPH and the SCR2 mutant, respectively.As discussed above, in addition to the inactivation of subunits of C3 convertases (C3b and C4b), sCCPH also regulates C3 convertases by accelerating their decay. It possesses considerable DAA for the CP/lectin pathway C3 convertase (C4b,2a) and a poor decay activity for the AP C3 convertase (C3b,Bb). Thus, we next examined the DAAs of the various sCCPH mutants to map the domains required for this function. To measure the CP C3 convertase decay activity, the C4b,2a enzyme was formed on sheep erythrocytes and allowed to decay in the presence of various mutants. The remaining enzyme activity was then measured by incubating the reaction mixture with EDTA sera (a source of C3 to C9) and measuring hemolysis. Apart from sCCPH, mutants SCR1-3, -1-2, and -2-3 showed substantial DAA for the CP C3 convertase (Fig. ​(Fig.4).4). These data suggested that SCR1-3 is primarily responsible for this activity. On a molar basis, SCR1-3 was 1.6-fold less efficient than sCCPH. Because both SCR1-2 and SCR2-3 possessed the decay activity, it was likely that similar to the cofactor activities, a single SCR domain of sCCPH might also possess the DAA for the CP C3 convertase. Hence, we also assessed the DAAs of the single-domain mutants. Interestingly again, SCR2 was the only single domain that distinctly displayed CP DAA (Fig. ​(Fig.4);4); however, on a molar basis, it was 26-fold less active than sCCPH. Previous data on the involvement of SCR domains in decay acceleration of CP C3 convertase in human RCA proteins (decay-accelerating factor, complement receptor 1, and C4b-binding protein) (3, 5, 20) and viral RCA homologs (Kaposica and VCP) (27, 30) have shown that a minimum of two or three consecutive domains are necessary for the activity. Thus, sCCPH is the only prototype to date in which a single SCR is adequate to impart the CP DAA.Open in a separate windowFIG. 4.Analysis of CP and AP C3 convertase DAAs of sCCPH and its mutants. (Upper panel) The CP C3 convertase C4b,2a was formed on antibody-coated sheep erythrocytes (EA) by sequentially incubating them with human C1, C4, and C2 (Calbiochem). The C3 convertase on the cells was then allowed to decay by incubating EA-C4b,2a with various concentrations of sCCPH or its mutants for 5 min at 22°C, and the activity of the remaining enzyme was assessed by measuring the cell lysis following incubation for 30 min at 37°C with Guinea pig sera containing 40 mM EDTA (27, 32). (Lower panel) The AP C3 convertase C3b,Bb was formed on sheep erythrocytes (ES) by incubating them with human C3 (Calbiochem) and factors B and D in the presence of NiCl₂. The C3 convertase on the cells was then allowed to decay by incubating ES-C3b,Bb with various concentrations of sCCPH or its mutants for 10 min at 37°C, and the activity of the remaining enzyme was assessed by measuring the cell lysis following incubation with EDTA-sera for 30 min at 37°C (35, 37). The data obtained were normalized by considering the lysis that occurred in the absence of an inhibitor as 100% lysis.Although sCCPH is known to possess limited AP C3 convertase DAA, we sought to determine whether this limited activity is localized in a specific region or the full-length protein. To measure the AP DAA, the C3 convertase C3b,Bb was formed on the sheep erythrocytes and incubated with sCCPH or with each of its deletion mutants. The decay of the AP C3 convertase was assessed by adding EDTA sera and measuring hemolysis. Although the full-length protein displayed a limited AP C3 convertase, none of the deletion mutants exhibited any activity (Fig. ​(Fig.44).Inactivation of C3 convertases by the RCA proteins, owing to their cofactor and decay activities, requires interaction of these proteins with C3b and C4b. The ligand binding activity of the RCA proteins, however, does not always correlate with their cofactor and decay activities (12, 34), as apart from ligand binding, cofactor activity involves interaction of the RCA protein with factor I (40), and decay activity involves interaction of the RCA protein with C2a or Bb (22, 25). In order to determine whether cofactor and decay activity data of sCCPH and the various mutants correlate with the ligand binding data, we measured binding of these proteins to C3b and C4b by using a surface plasmon resonance-based assay (38). As observed earlier (38), sCCPH displayed higher affinity for C4b than for C3b (Fig. ​(Fig.55 and Table ​Table1).1). When we measured binding of various deletion mutants to C3b and C4b, only SCR2-4 showed binding to C3b, and SCR1-3 showed binding to C4b (Fig. ​(Fig.5).5). However, there were reductions of about 16- and 14-fold in the affinities of these deletion mutants for C3b and C4b, respectively, compared to that for sCCPH (Table ​(Table1),1), suggesting that all the four domains contribute to binding to C3b and C4b. Because most of the deletion mutants that displayed complement regulatory activities possessed negligible binding to C3b and C4b, it is clear that binding of the mutants does not correlate with their cofactor and decay activities. It is likely that during cofactor activity, interaction of the mutants with C3b and C4b is stabilized by the interaction of factor I with C3b/C4b and the mutants. Similarly, during DAAs, the mutants may possess better affinity for the convertases than their subunits C3b and C4b. Consistent with this argument, decay-accelerating factor has previously been shown to bind to CP C3 convertase with 1,000-fold higher affinity than to C4b (33).Open in a separate windowFIG. 5.Binding of sCCPH and its mutants to C3b and C4b. Binding was determined by a surface plasmon resonance-based assay (38). Sensograms were generated by immobilizing biotinylated C3b (1,200 response units [RUs]) and C4b (940 RUs) on streptavidin chips (Sensor Chip SA; Biacore AB; additional RUs of C3b [∼6,000 RUs] were deposited by forming AP C3 convertase on the chip and flowing native C3 [14]) and injecting sCCPH or its mutants in PBS-T (10 mM sodium phosphate and 145 mM NaCl, pH 7.4, containing 0.05% Tween 20) over the chip. Flow cells immobilized with bovine serum albumin-biotin (Sigma) served as control flow cells. (Left panels) Binding of sCCPH and its various mutants to C3b (top) and C4b (bottom). The sensograms were generated by injecting 500 nM and 2 μM of sCCPH and its various mutants over C3b and C4b chips, respectively. (Middle panels) Sensogram overlay for the interaction between sCCPH and C3b (top) or sCCPH and C4b (bottom). (Right panels) Sensogram overlay for the interaction between SCR2-4 and C3b (top) and SCR1-3 and C4b (bottom). The concentrations of proteins injected are indicated at the right of the sensograms. The solid lines in the top middle and top right panels represent the global fitting of the data to a 1:1 Langmuir binding model with a drifting baseline (A + B ↔ AB; Biaevaluation 4.1). The small arrows in the bottom middle and right panels indicate the time points used for evaluating the steady-state affinity data.

TABLE 1.

Kinetic and affinity data for the interactions of sCCPH and the deletion mutants with human complement proteins C3b and C4b^a

Implantation of the Micra transcatheter pacing system: A single center North India experience

BackgroundThe leadless pacemaking transcatheter system, Micra, is a miniaturized, single-chamber pacemaker system. We report herein our experience with implantation of the Micra TPS system.ObjectiveThe current study was conducted to evaluate the safety and efficacy of the leadless Micra Transcatheter Pacemaker System (Medtronic).Research design and methodsThis was a prospective single centre nonrandomized study without controls. A transcatheter pacemaker was implanted in patients who had guideline based indications for ventricular pacing. 28 subjects were screened based on the selection criteria. Mica TPS was implanted. Parameters assessed were: duration of procedure (from femoral vein puncture to venous access closure), fluoroscopy time, number of device repositions, periprocedural electrical measurements (sensing, threshold and impedance) and in-hospital, intermediate to long term adverse events related to procedure.Result and conclusions: The device was successfully implanted in 28 subjects. The mean intraoperative sensing value was 9.04 ± 1.5 mV and the impedance was 766.89 ± 213.9 Ω. At discharge from hospital, those values were 13.2 ± 15.83 mV and 855 ± 111.7, respectively. The recommended pacing threshold value as achieved in all subjects was 0.78 V, i.e. ≤ 1 V at 0.24 ms. There was no adverse event or complications reported for any of the subjects. Mean time from hospitalization to discharge was 1.5 days. Implantation of leadless pacemakers is feasible, safe and provides advantages over the conventional system.     

High Proportion of Intestinal Colonization with Successful Epidemic Clones of ESBL-Producing Enterobacteriaceae in a Neonatal Intensive Care Unit in Ecuador

Background and Aims

Neonatal infections caused by Extended-spectrum beta-lactamase (ESBL)-producing bacteria are associated with increased morbidity and mortality. No data are available on neonatal colonization with ESBL-producing bacteria in Ecuador. The aim of this study was to determine the proportion of intestinal colonization with ESBL-producing Enterobacteriaceae, their resistance pattern and risk factors of colonization in a neonatal intensive care unit in Ecuador.

Methods

During a three month period, stool specimens were collected every two weeks from hospitalized neonates. Species identification and susceptibility testing were performed with Vitek2, epidemiologic typing with automated repetitive PCR. Associations between groups were analyzed using the Pearson _X ² test and Fisher exact test. A forward step logistic regression model identified significant predictors for colonization.

Results

Fifty-six percent of the neonates were colonized with ESBL-producing Enterobacteriaceae. Length of stay longer than 20 days and enteral feeding with a combination of breastfeeding and formula feeding were significantly associated with ESBL-colonization. The strains found were E. coli (EC, 89%) and K. pneumoniae (KP, 11%) and epidemiological typing divided these isolates in two major clusters. All EC and KP had bla _CTX-M group 1 except for a unique EC isolate that had bla _CTX-M group 9. Multi-locus sequence typing performed on the K. pneumoniae strains showed that the strains belonged to ST855 and ST897. The two detected STs belong to two different epidemic clonal complexes (CC), CC11 and CC14, which previously have been associated with dissemination of carbapenemases. None of the E. coli strains belonged to the epidemic ST 131 clone.

Conclusions

More than half of the neonates were colonized with ESBL-producing Enterobacteriaceae where the main risk factor for colonization was length of hospital stay. Two of the isolated clones were epidemic and known to disseminate carbapenemases. The results underline the necessity for improved surveillance and infection control in this context.     

Nitrification Rates in the Baltic Sea: Comparison of Three Isotope Techniques

Simultaneous measurements of nitrification in the Baltic Sea were made at 10- to 30-m intervals in the months of June and November by three isotope techniques: [¹⁵N]nitrate dilution, N-serve sensitive [¹⁴C]bicarbonate incorporation, and [¹⁵N]ammonium oxidation to nitrite and nitrate. Nitrification rates of 1 to 280 nmol liter⁻¹ day⁻¹ were recorded, and each method showed that the highest rates of nitrification occurred below the halocline. Even in the presence of ammonium, dark incubations of mixed layer (above ca. 50 m) waters never yielded nitrification rates exceeding 45 nmol liter⁻¹ day⁻¹. The rates measured by the ammonium oxidation method were two- to sevenfold greater than those obtained by ¹⁴C incorporation or ¹⁵N dilution. The merits of each technique are discussed, and it is suggested that the [¹⁵N]ammonium oxidation method should be used in conjunction with the [¹⁴C]bicarbonate incorporation method.     

Membranes of class IIa bacteriocin-resistant Listeria monocytogenes cells contain increased levels of desaturated and short-acyl-chain phosphatidylglycerols

A major concern in the use of class IIa bacteriocins as food preservatives is the well-documented resistance development in target Listeria strains. We studied the relationship between leucocin A, a class IIa bacteriocin, and the composition of the major phospholipid, phosphatidylglycerol (PG), in membranes of both sensitive and resistant L. monocytogenes strains. Two wild-type strains, L. monocytogenes B73 and 412, two spontaneous mutants of L. monocytogenes B73 with intermediate resistance to leucocin A (+/-2.4 and +/-4 times the 50% inhibitory concentrations [IC50] for sensitive strains), and two highly resistant mutants of each of the wild-type strains (>500 times the IC50 for sensitive strains) were analyzed. Electrospray mass spectrometry analysis showed an increase in the ratios of unsaturated to saturated and short- to long-acyl-chain species of PG in all the resistant L. monocytogenes strains in our study, although their sensitivities to leucocin A were significantly different. This alteration in membrane phospholipids toward PGs containing shorter, unsaturated acyl chains suggests that resistant strains have cells with a more fluid membrane. The presence of this phenomenon in a strain (L. monocytogenes 412P) which is resistant to both leucocin A and pediocin PA-1 may indicate a link between membrane composition and class IIa bacteriocin resistance in some L. monocytogenes strains. Treatment of strains with sterculic acid methyl ester (SME), a desaturase inhibitor, resulted in significant changes in the leucocin A sensitivity of the intermediate-resistance strains but no changes in the sensitivity of highly resistant strains. There was, however, a decrease in the amount of unsaturated and short-acyl-chain PGs after treatment with SME in one of the intermediate and both of the highly resistant strains, but the opposite effect was observed for the sensitive strains. It appears, therefore, that membrane adaptation may be part of a resistance mechanism but that several resistance mechanisms may contribute to a resistance phenotype and that levels of resistance vary according to the type of mechanisms present.