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Dimethylsulfoniopropionate (DMSP), an abundant osmoprotectant found in marine algae and salt marsh cordgrass, can be metabolized to dimethyl sulfide (DMS) and acrylate by microbes having the enzyme DMSP lyase. A suite of DMS-producing bacteria isolated from a salt marsh and adjacent estuarine water on DMSP agar plates differed markedly from the pelagic strains currently in culture. While many of the salt marsh and estuarine isolates produced DMS and methanethiol from methionine and dimethyl sulfoxide, none appeared to be capable of producing both methanethiol and DMS from DMSP. DMSP, and its degradation products acrylate and β-hydroxypropionate but not methyl-3-mecaptopropionate or 3-mercaptopropionate, served as a carbon source for the growth of all the α- and β- but only some of the γ-proteobacterium isolates. Phylogenetic analysis of 16S rRNA gene sequences showed that all of the isolates were in the group Proteobacteria, with most of them belonging to the α and γ subclasses. Only one isolate was identified as a β-proteobacterium, and it had >98% 16S rRNA sequence homology with a terrestrial species of Alcaligenes faecalis. Although bacterial population analysis based on culturability has its limitations, bacteria from the α and γ subclasses of the Proteobacteria were the dominant DMS producers isolated from salt marsh sediments and estuaries, with the γ subclass representing 80% of the isolates. The α-proteobacterium isolates were all in the Roseobactersubgroup, while many of the γ-proteobacteria were closely related to the pseudomonads; others were phylogenetically related toMarinomonas, Psychrobacter, or Vibrio species. These data suggest that DMSP cleavage to DMS and acrylate is a characteristic widely distributed among different phylotypes in the salt marsh-estuarine ecosystem.
Dimethyl sulfide (DMS)-producing bacteria play an important but as yet unquantified role in the biogenic transfer of sulfur from the ocean to the atmosphere (2, 25, 30). DMS production results from the enzymatic degradation of dimethylsulfoniopropionate (DMSP) (9, 28, 43), an osmoprotectant (13, 14, 22, 47) produced and stored by marine phytoplankton, macroalgae, cyanobacteria, and coastal vascular plants (e.g., Spartina alterniflora) (7, 8, 23, 38, 50). When these organisms senesce and decay or phytoplankton are grazed upon by zooplankton, the intracellular DMSP is released into the water column or sediment (9, 35, 53), where it can be used as a carbon and energy source for the bacterial community (25, 49). The enzymatic degradation of DMSP to DMS and acrylate has been observed in marine and estuarine bacteria (11, 25, 31), fungi (4), and algae (6, 22, 41). The enzyme responsible, DMSP lyase, has been purified from several marine bacteria (11, 12, 48).
Understanding the factors controlling DMS production in the marine environment relies on knowing the abundance, phenotypic diversity, and physiology of the microbes involved in this process. As there is not a functional probe available to quantitate and identify DMS-producing microbes in environmental samples, studies of DMS production have been limited to isolation and characterization of DMS-producing strains (11, 17, 21, 25, 31, 48, 52). The limitations in culturing marine bacteria have long been known (see reference 36), so it is probable that only a small percentage of DMS producers have been isolated. In recent studies of the diversity of DMS-producing bacteria from oceanic and estuarine waters it was found that all the isolates belonging to the Roseobacter subgroup of the α subdivision of the Proteobacteria were DMS producers (17, 31). Since the Roseobacter group accounted for almost 30% of the 16S ribosomal DNA (rDNA) in coastal and estuarine water (19), it suggested that this group of DMS-producing bacteria is quite common in the marine environment (17). However, other DMS-producing phylotypes from the marine environment belonging to the β (11), γ (21, 31), and δ (48) subdivisions of theProteobacteria have also been identified, but their prominence remains unknown.
S. alterniflora-dominated salt marshes represent another marine ecosystem where DMS production occurs. In fact the rates per unit area of salt marsh are much higher than those of coastal and ocean water (42) and may support a unique assemblage of DMS producers. The focus of this research was to assess the phenotypic and phylogenetic diversity of DMS-producing bacteria from the marsh and adjacent estuarine waters. The culturable DMS producers from these sources obtained by plating dilutions on minimal DMSP medium as described below belonged predominantly to the γ subdivision and, to a lesser extent, the α subdivision of Proteobacteria.
Isolation of DMS-producing bacteria.Surface sediment and estuarine water samples (salinity, ca. 35 ppt) were obtained from North Inlet, Georgetown, S.C. DMS-producing bacteria were obtained by plating serial dilutions of estuarine water or sediment slurries directly on modified basal salts (3) or f/2 medium supplemented with DMSP (1 mM). The latter is a minimal, seawater-based medium (20). Additional isolates were obtained from 5-day enrichment cultures grown in f/2-DMSP medium, followed by plating of serial dilutions onto f/2-DMSP agar plates. In both cases the dilutions were sufficient to yield 30 to 100 colonies when 100 μl was spread on plates of marine broth (Bacto). After several days of growth, colonies with diverse morphologies were selected for further testing after purification.
Production of volatile organosulfur compounds.Isolates were grown in either marine or tryptic soy broth (5-ml cultures) for 24 h, at which time the cultures reached maximum turbidity. The cultures were harvested by centrifugation for 30 s, the pellet was resuspended in an equal volume of half-strength seawater, and 1-ml aliquots were placed in 14.5-ml glass serum bottles. Organosulfur compounds (DMSP, methionine, dimethyl sulfoxide, methyl-3-mercaptopropionate [MMPA], or 3-mercaptopropionate [MPA]) were added at 1 mM concentrations from 150 mM neutralized stock solutions, and the cultures were incubated at 23°C for 3 days on a rotary shaker (100 rpm). DMS, hydrogen sulfide, and methanethiol were analyzed at 24-h intervals by gas chromatography as described previously (11).
Growth substrates.Overnight cultures grown in minimal medium containing 0.05% yeast extract were used to inoculate tubes of minimal media (either f/2 or modified basal salts) containing either DMSP, acrylate, β-hydroxypropionate (β-HP), glycine betaine (GBT), MPA, or MMPA as the carbon and energy source. All the carbon sources were added to a final concentration of 5 mM from a filter-sterilized stock solution to autoclaved media. Cultures were incubated on a roll shaker at 30°C, and growth was monitored with a Klett spectrophotometer.
Isolation of chromosomal DNA and PCR amplification of the 16S rRNA gene.Isolates were grown on either tryptic soy or marine broth overnight and then subcultured into 50 ml of the same medium for 5 h at 37°C on a rotary shaker (150 rpm). Cells were harvested by centrifugation (6,000 × g for 10 min at 4°C), and chromosomal DNA was isolated from cell pellets using a standard procedure (33). Purified DNA from the DMS-producing isolates was used as the template in a PCR to amplify the 16S rRNA coding regions. Bacterium-specific primers (GM3F and GM4R) (see Table1) were used to amplify the 16S rRNA gene. PCR mixtures contained the following per 50 μl of reaction mixture: ca. 10 ng of template, 45 pmol of each primer (GM3F and GM4R), 10 μmol of each deoxyribonucleoside triphosphate, 5 μl of 10× PCR buffer, 1.5 mM MgCl2, and TaqBead Hot Start polymerase (Promega Corp., Madison, Wis.). PCR amplifications were performed using an Eppendorf Mastercycler gradient. To reduce the chance of spurious by-product formation and increase the specificity of the amplification, a "touchdown" PCR was performed (15). Denaturation of the DNA was carried out at 94°C for 30 s, while the annealing temperature was set at 50°C, which was 10°C above the expected annealing temperature. The temperature was decreased by 1°C every second cycle until a touchdown of 40°C was achieved, at which temperature 10 additional cycles were carried out. Elongation was carried out at 72°C for 2 min, with a total of 30 cycles.
PCR products (5 μl) were analyzed by electrophoresis on a 0.8% (wt/vol) agarose gel to ensure the correct size of the amplified DNA. Products were then purified using the QIAquick PCR purification kit (Qiagen Inc., Valencia, Calif.) and ligated into the pGem-T vector (Promega), and the hybrid vector was used to transformEscherichia coli JM109 competent cells (Promega). PCR purification, ligation, and transformation were performed using the manufacturer's instructions. Recombinants were selected on tryptic soy agar plates supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside, isopropyl-β-d-thiogalactopyranoside, and 100 μg of ampicillin · ml−1. Transformants harboring the hybrid vector (white colonies) were subsequently patched onto tryptic soy agar plates supplemented with 100 μg of ampicillin · ml−1 and analyzed for the 16S rRNA gene plasmid insert by a colony PCR procedure using the GM3F and GM4R primers. One positive clone harboring the 16S rRNA gene insert from each DMS-producing isolate was chosen for partial sequence analysis.
Plasmid isolation and 16S rRNA gene sequencing.Plasmids were purified from transformants grown overnight in tryptic soy Broth (5 ml) supplemented with 100 μg of ampicillin · ml−1 using the alkaline pH method (5). The 16S rRNA gene insert was amplified using M13 primers that are complementary to the plasmid sequences flanking the insert. PCR conditions were as follows: initial denaturation was carried out at 95°C for 3 min followed by 36 cycles of denaturation at 95°C for 20 s, annealing at 50°C for 20 s, and elongation at 72°C for 1.5 min. PCR products were purified using the QIAquick PCR purification kit. Sequences spanning the V3 and V5 hypervariable regions of the rRNA gene (35a) were sequenced on both strands using primers DY23 and DY24 (Table 1). Primer DY23 corresponds to positions 341 to 357 of the E. coli numbering system, and DY24 corresponds to positions 907 to 926 of the E. coli numbering system. The sequences were determined using the ABI Prism BigDye terminator cycle sequencing kit and ABI Prism 377 sequencer according to the manufacturer's directions. Complementary sequences were aligned using the Sequencer program (Gene Codes Corp., Ann Arbor, Mich.).
Phylogenetic analysis of the 16S rDNA sequences.To determine the nearest phylogenetic neighbors, each partial sequence of the cloned 16S rDNA genes was compared to the National Center for Biotechnology Information GenBank database using a homology search tool, BLAST (1). For phylogenetic trees, sequences were aligned using the CLUSTAL W multiple sequence alignments program (version 1.81) (46). The alignment was performed using several bacteria that were closely related to the unknown organisms based on information obtained using the BLAST search and unrelated phyla as outgroups. Additional details are given in the figure legends.
Nucleotide sequence accession numbers.The 16S rDNA sequences of the isolates from the North Inlet, S.C., salt marsh and estuary and their GenBank accession numbers are as follows: JA6,AF296133 ; JA33, AF296134 ; JA41, AF296135 ; JA22, AF296136 ; JA3,AF296137 ; JA1 AF296139 ; JA14, AF296139 ; JA11, AF296140 ; JA17, AF296141 ; JA42, AF296142 ; JA31, AF296143 ; JA32, AF296144 ; JA23, AF296145 ; JA27, AF296146 ; JA13, AF296147 ; JA35, AF296148 ; JA45, AF296149 ; JA29, AF296150 ; JA30, AF296151 ; JA20, AF296152 ; JA9, AF296153 ; JA19,AF296154 ; JA34, AF296155 ; JA16, AF296156 ; and JA25, AF296157 . Isolate M3A was identified earlier as Alcaligenes faecalisstrain M3A (AF155147 ).
Reference 16S rDNA sequences from the GenBank database used in the phylogenetic analyses were as follows, by group.
(i) α-Proteobacteria. Rhodospirillum rubrum, X87278 ; SAR11, X52172 ; Agrobacterium tumefaciens, D13943 ; Paracoccus denitrificans, X69159 ;Rhodobacter sphaeroides, D16424 ; Roseobacter sp. strain DSS-1, AF098492 ; Sulfitobacter sp. strain DSS-2,AF098490 ; Silicibacter sp. strain DSS-3, AF098491 ;Marinosulfonomonas methylotrophus, U62894 ;Sulfitobacter mediterraneus, Y17387 ; Roseobacter denitrificans, M96746 ; Roseobacter gallaeciensis,Y13244 ; Antarctobacter heliothermus, Y11552 ;Ruegeria atlantica, AF124521 ; Silicibacter lacuscaerulensis, U77644 ; Sagittula stellata E-37,U58356 ; strain LFR (DMSP-degrading bacterium), L15345 ; and uncultured marine bacterium D033, AF177566 . Bacillus subtilis,AB018486 , was used as an outgroup.
(ii) β-Proteobacteria. Alcaligenes faecalis strain M3A (ATCC 700596), AF155147 ;Alcaligenes faecalis, D88008 ; Alcaligenes strain 05-36, X86580 ; Alcaligenes defragrans, AJ005450 ;Ralstonia eutropha, Y10825 ; Bordetella parapertussis, U04949 ; Bordetella pertussis, AF142327 ;Bordetella hinzii, AF177667 ; Bordetella avium,AF177666 ; Achromobacter xylosoxidans, AF225979 ; andAchromobacter denitrificans, AF232712 .
(iii) γ-Proteobacteria. Alteromonas macleodii, X82145 ; Pseudomonas doudoroffii,AB021371 ; E. coli, J01859 ; Vibrio mytili, X99761 ;Acinetobacter junii, X81658 ; Acinetobacter anitratus, U10874 ; Moraxella lacunata, AF005171 ;Psychrobacter immobilis, U39399 ; Psychrobacter glacincola, PGU85876; Psychrobacter pacificensis,AB016059 ; Neptunomonas napthovorans, AF053734 ;Marinobacterium georgiense, U58339 ; Pseudomonas stanieri, X92176 ; Marinomonas vaga, X67025 ;Marinomonas protea, AJ238597 ; Pseudomonas fluorescens, AF228367 ; Pseudomonas fulva, D84015 ;Pseudomonas stutzeri, AJ288148 ; Pseudomonas aeruginosa, AF237678 ; and Pseudomonas putida, U70977 .
Phospholipid analysis.Certain microbes were also identified by phospholipid fatty acid analysis using the MIDI microbial identification system (Newark, Del.) (40).
Metabolism of DMSP and its metabolites.Several hundred colonies that grew rapidly on DMSP-containing plates were isolated from estuarine water samples and DMSP enrichment cultures inoculated with salt marsh surface sediment. All were capable of DMS cleavage from DMSP. Twenty-five isolates were chosen from this group based on their differences in colony morphology; all proved to be aerobic, gram-negative rods. The initial kinetics of DMS production from DMSP showed considerable variability among the isolates in carrying out this reaction (Fig. 1). The DMSP added at time zero served as both the inducer of the DMSP lyase and its substrate, and variability among the isolates is seen in both DMSP-dependent processes. One of the isolates (JA27) was able to metabolize DMSP to DMS with no apparent lag period, suggesting that the DMSP lyase might be constitutively expressed, a characteristic never before seen in DMSP utilizers.
DMSP and its degradation products DMS, acrylate, β-HP, MMPA, and MPA, and a DMSP analog, GBT, are all potentially important carbon and energy sources for marine bacteria, and as such the DMS-producing isolates were tested for their ability to grow on these molecules (Table2). DMSP served as a growth substrate for all isolates; however, acrylate, a product of DMS cleavage, served as a growth substrate for some but not all. MMPA and MPA, the products of DMSP demethylation (45), were all ineffective as was DMS (data not shown). The metabolism of acrylate to β-HP and the utilization of the latter as a carbon and energy source was observed in two DMS producers, A. faecalis M3A (3) and strain LFR (J. H. Ansede, P. J. Pellechia, and D. C. Yoch, unpublished data). When β-HP was tested as a growth substrate for the isolates, the pattern was identical only among the α-proteobacteria to that of its precursor, acrylate (Table 2). GBT, an abundant quaternary ammonium compound found among DMSP-producing marine algae and plants and an important carbon and energy source for marine bacteria (29), supported growth of most of the DMS-producing isolates. Among the phylotypes, the α-proteobacteria were the most versatile in using these substrates for growth. None of these isolates grew on DMS, nor did they degrade it, as it did not disappear from the gas phase once produced by the action of DMSP lyase (Fig. 1). One isolate (JA25), although isolated on a DMSP plate, could not subsequently be cultured on DMSP in liquid medium and did not produce DMS from DMSP. It did, however, produce methanethiol from DMSP. Although an α-proteobacterium, it was not closely related to the DMS producers (Fig.2).
The isolates were also characterized for their ability to volatilize organosulfur compounds found in marine environments (Table3). Many were able to reduce DMSO to DMS (probably via DMSO reductase activity) and metabolize methionine to methanethiol. None of the DMS-producing isolates were able to metabolize MMPA to MPA or methanethiol, or MPA to hydrogen sulfide. Dimethylsulfonioacetate, a DMSP analog, was ineffective as a substrate for DMS volatilization (data not shown), and dimethyl disulfide and methanethiol were not degraded by any of the isolates (data not shown). These results must be viewed with caution because of the high concentrations tested.
Phylogenetic analysis.The 16S rRNA gene sequences used for phylogenetic analysis of the DMS-producing salt marsh and estuarine isolates were approximately 500 nucleotides in length and spanned the V3 and V5 hypervariable regions of the gene. These sequences were submitted for BLAST analysis (1) and subjected to phylogenetic analyses by using the PAUP program to gain an understanding of their relationship to each other and their phylogenetic positions relative to known sequences. All sequences submitted showed ≥93% similarity to those found in the GenBank database (32), and while some were >99% similar, none were exactly identical to the sequences of cultured organisms or environmental clones in the database. The closest match was isolate JA3, which had a 99.7% partial-sequence similarity to P. putida. Phylogenetic analysis identified all of the DMS-producing isolates as members of the class Proteobacteria, most of which were in the γ subclass.
The phylogenetic tree of the isolates belonging to the α subgroup of the Proteobacteria is presented in Fig. 2. Distance and maximum likelihood analyses of the partial 16S rRNA gene sequences produced similar results. All strains capable of producing DMS, whether from this study (prefaced by "JA") or the literature, are indicated in bold. All of the DMS-producing α-proteobacterium isolates were in the Roseobacter subgroup of the Rhodobactergroup. Isolates JA13, JA20, and JA34 formed a tight cluster that grouped close to another marine DMS producer, Sagitulla stellata (17). Isolate JA6 (not shown on the tree) was placed close to R. sphaeroides and P. denitrificans, showing 96% sequence homology to the latter. Isolate JA25, which did not group near any of the DMS producers, was the strain that did not grow on DMSP or produce DMS but did produce methanethiol from it. This is apparently unusual as all methanethiol producers isolated to date also produce DMS from DMSP (see reference26).
Of all the DMS-producing bacteria isolated over a 5-year period from the salt marsh and estuarine water, only one isolate,Alcaligenes strain M3A, identified by its phenotypic characteristics (11) and phospholipid fatty acid content (data not shown), belonged to the β-proteobacteria. Analysis of 1,536 bp of the 16S rRNA gene indicated more specifically that it was anA. faecalis subspecies having 99.3% sequence similarity to the type strain, ATCC 8750. The type strain, a terrestrial isolate, does not degrade DMSP (data not shown). The phylogenetic tree shows the relationship of strain M3A to its closest β-proteobacterial relatives (Fig. 3).
The most abundant group of the salt marsh and estuarine DMS-producing isolates (18 of 24) was affiliated with the γ subdivision ofProteobacteria (see the Fig. 4tree). They were widely dispersed among various taxa and appeared to group into five clusters, with numerous isolates clustering near the pseudomonads and the genera Marinomonas, Psychrobacter, and Vibrio.
Bacteria play an important role in the cycling of sulfur within the marine environment, and the catalysis of DMSP to DMS is an important, well-established part of this cycle. Therefore, knowing the diversity of the population involved is essential to understanding the global sulfur cycle. Prior to this study there were about a dozen DMS-producing isolates whose physiology and phylogeny had been analyzed, most of which were isolated from marine waters. In addition to single isolates belonging to the β, γ, and δ subdivisions of the Proteobacteria, numerous isolates of the α subdivision of the Roseobacter subgroup were obtained from either open ocean or coastal waters which also proved to be DMS producers. The latter observation led to the suggestion that this class of bacteria represented a prominent lineage of DMS producers and "play an important role in the marine sulfur cycle" (17). While α-proteobacteria may also prove to play an important role in the sediments of a Spartina-dominated salt marsh and adjacent estuarine water, the phylogenetic pattern seen here on examining 16S rDNA sequences of DMS producers isolated suggested that γ-proteobacteria may be even more important in this ecosystem. Of the culturable DMS producers deemed to be unique based on phenotypic characteristics, partial 16S rDNA sequence analysis showed that 80% were γ-proteobacteria and <20% were α-proteobacteria (cf. Fig. 2 and 4). Although not quantitative, and no doubt dependent on the isolation technique employed, this study has extended the DMS-producing characteristic into new phylogenetic territory.
The apparent predominance of γ-proteobacterial DMS producers in these North Inlet tidal creeks contrasts with analysis of community DNA of microbes that attach to submerged surfaces in that 85% of the clones sequenced were affiliated with the Roseobactersubgroup of the α-proteobacteria (10). One of those clones, DO33, showed 98% sequence similarity to members of a clade of DMS producers (JA13, JA20, and JA34) isolated from the same tidal creek and adjacent salt marsh, and all were related to another DMS producer,S. stellata E-37 (Fig. 2). A somewhat unrelated isolate, JA19, is closely related to another nearshore DMS producer,Silicibacter sp. strain DSS-3 (17).
The β-proteobacteria are much less common in both estuarine (10) and oceanic water (18, 24, 37, 43), and similar results are reported here. Only one DMS-producing β-proteobacterium, Alcaligenes strain M3A, whose isolation was previously reported (11), was isolated from the salt marsh in numerous samplings of this ecosystem over a number of years. Analysis of full-length 16S rDNA showed strain M3A to be a subspecies of A. faecalis, apparently the first to be isolated from the marine environment. It appears to be quite common on the marsh surface, as microbes of identical phenotypes (25 carbon utilization abilities were assayed) were isolated many times. It is easily recognizable by its diffusable yellow pigment on agar plates, a characteristic not produced by the non-DMS-producing terrestrial A. faecalis strains. The isolated pigment had an absorbance maximum between 390 and 490 nm (data not shown). Not only does the type strain of A. faecalis (ATCC 8750) not have the pigment, it does not metabolize DMSP to DMS, nor could it grow on acrylate or β-HP, products of the DMSP degradation pathway. The isolation of other species of DMS-producing β-proteobacteria may require different isolation techniques.
The majority of the DMS-producing isolates from the marsh whose rDNA was sequenced proved to be γ-proteobacteria (Fig. 4). Several isolates (JA3, JA14, JA11, and JA33) were closely related to cultured pseudomonads. Isolate JA3, in fact, was positively identified as P. putida by both its phospholipid fatty acid composition (data not shown) and its 16S rRNA gene, which showed >99% sequence similarity to that of P. putida. Four isolates (JA42, JA29, JA30, and JA22) form a cluster that is closely related to several Marinomonas species. Two other isolates (JA17 and JA23) did not group closely to other marine bacteria, and therefore it may be of interest to further characterize them. Isolate JA9 was found closest to N. napthovoranson the tree. Several of the DMS-producing isolates (JA31, JA32, JA45, and JA16) were found to cluster among a group of psychrophilic bacteria, showing 97 to 98% sequence similarity toP. pacificensis, a deep seawater isolate from the Japan Trench (34). Two other isolates, JA1 and JA35, were closely related to the marine vibrio V. mytili. The sequence differences between these two isolates are due mostly to transitions, which suggests that they could be more closely related than the tree indicates. Finally, isolate JA27, which shows no apparent lag prior to producing DMS from added DMSP, is related to the marine DMS-producing bacterium P. doudoroffii.
The salt marsh and estuarine isolates were characterized as to their ability to metabolize various organosulfur compounds and DMSP degradation products that occur as sulfur cycle intermediates in the marine environment. Anoxic salt marsh sediments amended withdl-methionine were previously shown to yield methanethiol as the major, volatile organosulfur product (28). Many of the aerobic isolates from this study that produce DMS from DMSP also metabolized methionine to methanethiol. Presumably both methionine and DMSP are released into the sediment pore water and water column during periods of phytoplankton senescence and therefore would serve as both a carbon and energy source for the bacterial community.
An alternative route of DMSP metabolism is its demethylation to MMPA, which may then be subsequently degraded to MPA or methanethiol (27, 44, 51). This demethylation mechanism has been observed in both oxic and anoxic marine sediments and in a number of DMS-producing α-proteobacterium isolates (17). Interestingly, out of the several hundred DMS-producing isolates obtained from salt marshes and tidal creeks in this study, only one demethylating strain was isolated. Since DMSP demethylating strains apparently dominate in the marine environment, the isolation of mostly DMS producers reported here can be explained only if they grew more rapidly (24 to 48 h) on 1 mM DMSP agar plates. These results are, however, consistent with a report by Taylor and Gilchrist (44) who found that "enrichments with DMSP selected for bacteria that generated DMS, whereas MMPA enrichments selected organisms that produced methanethiol."
None of the DMS-producing isolates were able to metabolize DMSP to MMPA or MPA, as all the DMSP-sulfur could be accounted for as DMS (Fig. 2and similar data not shown), nor did any produce DMS and methanethiol concomitantly from DMSP. The latter characteristic was reported for several α-proteobacterium isolates (26). One isolate, JA19, that might have been expected to have demethylation activity did not, even though it showed 98% sequence similarity toSilicibacter sp. strain DSS-3, its nearest relative in the database, which has both lyase and demethylase activities (17).
The uptake of acrylate and β-HP seems to be phylotype related in that all α- and β-proteobacterium isolates were able to grow on DMSP, acrylate, and β-HP (Table 2). This correlates with the fact that representatives of these phylogenetic groups degrade DMSP or acrylate to β-HP on the cell surface and transport it into the cell (3, 54; Ansede, Pellechia, and Yoch, unpublished). The phenomenon of some isolates growing on DMSP but not on acrylate or β-HP may be explained if the concentration provided was inhibitory, which may also explain the lack of growth of numerous marine α-proteobacteria on 5 mM acrylate (17). P. doudoroffii and isolates that resemble it may not be able to grow on acrylate or β-HP simply because they cannot transport it. P. doudoroffiitransports DMSP into the cytosol where it is degraded to acrylate and DMS (54) and therefore may not have a need to transport the DMSP degradation products.
While the substrate utilization patterns reported here appear to have some relation to the phylogeny of the isolates, this pattern apparently does not hold up when the results from other studies are examined. For example, most the DMS-producing α-proteobacterium isolates of Gonzalez and Moran (19) did not grow on 5 mM acrylate (17, 26), unlike those reported here (Table 2).
To date, studies performed with natural populations of DMS producers have focused on culture-based techniques that may or may not reveal the extent of their actual diversity. However, it is recognized that culture-independent methods are needed to assess DMS-producing assemblages in their natural habitat. Unfortunately, at this time there are no probes available to identify the community involved in DMS production. Primers specific to conserved regions of the 16S rRNA genes that target only sulfate reducers, for example, have been developed and used for analyzing natural assemblages. However, DMS producers are widely dispersed among the Proteobacteria as shown here, and more recently were even found among the gram-positive bacteria (D. C. Yoch and R. N. Hardee, unpublished data). Therefore the development of primers to specifically target the 16S rRNA gene of DMS producers seems unlikely. Attempts are under way to clone the DMSP lyase gene so that a functional probe may be constructed. Such a probe could be used in identifying DMS-producing bacteria from natural populations, thereby eliminating the culture-based bias when accessing the diversity of the DMS-producing assemblage. Despite the limitations of using cultured bacteria to access their abundance in the natural environment, this study has nonetheless added to the accumulating knowledge of the diversity of the DMS-producing bacteria.
We thank Travis Glenn for his help and advice with the DNA sequencing and introduction to phylogenetic analysis of 16S rDNA.
This work was supported in part by a grant from the South Carolina Sea Grant Consortium.
- Received 29 September 2000.
- Accepted 4 January 2001.
- Copyright © 2001 American Society for Microbiology


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The chaos of battle and the vast scale of the assault thwarted attempts then -- and now -- to tally how many thousands were killed in the June 6, 1944, landings that sped Nazi Germany's defeat. Bodies disintegrated under bombs and shells. Soldiers drowned and disappeared. Company clerks who tallied casualties were killed. Records were lost. Historians say a definitive death toll will likely never be known. Even now, the Normandy soil for which soldiers fought so bitterly offers up new bodies. "Now and then, construction work unearths bones and skeletons from soldiers. This happens fairly often," said Fritz Kirchmeier, a spokesman for the German organization that tends the 80,000 graves for German soldiers in Normandy. Casualty estimates for Allied forces vary, but range from 2,500 to more than 5,000 dead on D-Day. Adding to the confusion is that D-Day books and histories often count wounded, missing and troops taken prisoner. On its Web site, the D-Day Museum in Portsmouth, England, says an estimated 2,500 Allied troops died. The U.S. Army Center of Military History in Washington, D.C., numbers 6,036 American casualties, including wounded and missing. The Heritage Foundation in Washington estimates 4,900 dead. "It's very difficult to get accurate figures. People get buried. Bodies disintegrate. Evidence of the deaths disappeared. People drowned," said John Keegan, author of "Six Armies in Normandy: From D-Day to the Liberation of Paris." More than 19,000 civilians in Normandy also died in Allied bombing before and after D-Day to soften up German defenses. And Allied air forces lost nearly 12,000 men in April and May 1944 in operations ahead of the invasion, the D-Day Museum says. Even as the ranks of veterans who survived the assault and the push into Germany thin with time, work on tallying the dead continues. Carol Tuckwiller, director of research at the National D-Day Memorial Foundation in Bedford, Va., has spent four years combing through government, military and cemetery records for names of Allied dead on D-Day. She hopes to have a figure by next year. "We feel like we're probably going to end up with a total of about 4,500 fatalities for both the Americans and Allied countries. Right now, we have about 4,200 names confirmed," she said. "Of course we realize we may never be 100 percent complete." In all, some 160,000 men invaded Nazi-occupied France in the first wave, in three paratroop and six infantry divisions, tank and commando units. The invasion fleet was the biggest armada in history, with more than 5,000 ships and landing craft. That more troops were not killed is testimony to the planning, training and overwhelming firepower of the Allies. Calculating German casualties is even harder. The D-Day Museum says the number is not known but is estimated at 4,000-9,000. Kirchmeier at the German graves commission said many records were destroyed in the Allied bombing of Berlin. D-Day marked only the start of the battle of Normandy, which claimed many more lives as troops fought in the region's hedgerows over the next three months. More than 425,000 Allied and German troops were killed, wounded or went missing, the D-Day Museum says. The American Cemetery overlooking Omaha Beach holds the remains of 9,383 servicemen and four women, their white gravestones a permanent reminder of war's terrible costs. "If you forget what happened here then you're never going to improve things. It's never going to get any better," said Donald Null, an 80-year-old veteran from Frederick, Md.
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- Elyria Police say woman attempts to stab pregnant woman
- Lorain police look for man who stole woman's purse
- Lorain County commissioners reject petition to vacate
- Train derails in Amherst Township
- Fire displaces family in Lorain
- More demolitions coming in late fall for Lorain homes
- Lorain Police Department wraps first month of Sweet Citations program


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November 4 is the last date to bid for the NTPC Mandsaur plant, which uses 40 numbers of 1250 KW grid-interactive power conditioning units (inverters).
Integrated power infrastructure offers an emerging investment opportunity in Asia as the region expands and adapts its energy mix to address sustainability and resilience goals. Narsingh Chaudhary and Mitesh Patel, of engineering, procurement and construction business Black & Veatch, tell pv magazine more.
The European Union and India's Ministry of New and Renewable Energy have jointly launched standard operation procedures and monitoring tool for Indian solar parks. While the procedures will help to ensure consistency of standards across solar parks, the online monitoring tool will allow better tracking of the solar parks development and operation.
Starting its journey as a department within Mahindra Group's solar EPC company Mahindra Susten, the newly formed comprehensive asset care company for renewable energy customers aims to reach a 20 GWp global portfolio by 2022 from 4 GWp currently.
The company's electric vehicle (EV) charging service combines physical components such as charging stations, swap stations and telematics systems, with virtual components like cloud service, analytics and artificial intelligence.
In a recent survey, DNV GL found that demand for blockchain-related skills in the solar sector is 50% higher than in other energy industries, while 33% of respondents said that the use of drones is having a significant impact on their businesses. However, the solar industry needs a common framework to help advance digitalization further.
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Tista' taqra bil- Malti.
The Apostolic Administrator for Gozo Bishop Mario Grech has urged the public to remain home and abide by the directives given by the health authorities in a new video which was published by the Diocese of Gozo.
He explained that one leaves their home and meets other people, one is not only putting at risk oneself but also others.
Bishop Mario Grech who was brought back to Malta on a repatriation flight and is currently under quarantine said that through this experience our mask has fallen off and we are showing our true vulnerable selves.
The Church in Malta and Gozo has closed all its churches and suspended mass in a bid to prevent the spread of the novel coronavirus. The outbreak has disrupted daily life, with schools closing down, many people asked to work from home and other measures being taken to help contain the spread.
Mgr Grech referred to a number of initiatives which took off as soon as restrictions were imposed. He said it is encouraging to see various individuals showing solidarity and assisting the elderly and most vulnerable individuals within our communities.
He warned against panic buying, saying that hoarding on essential products might lead to a situation were those without disposable income cannot buy beforehand and will end up being deprived of that product.
He urged the public to cherish life especially in such dark times.
Mgr Grech also showed his gratitude towards those on the frontline battling Covid-19, mentioning doctors, nurses, and healthcare workers as well as the disciplined forces. He also mentioned the priests whom he described as "spiritual doctors" that are accompanying the flock in this difficult moment.
He urged the public to pray and hope, ending his message that with God's help we shall overcome this moment.
If you want to be the first to receive the latest news on the coronavirus in Malta, download the Newsbook APP here.


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LAWRENCEVILLE — North Gwinnett's football team looked playoff-ready with a 59-14 win over host Mountain View in Friday night's regular-season finale.
The Bulldogs, seeded second in Region 8-AAAAAAA for the state playoffs, jumped ahead 22-7 after a quarter and 36-7 at halftime. They host Cherokee next Friday in the first round of state.
Marcus McFarlane had 10 rushes for 113 yards and two touchdowns, and Ethan Washington rushed eight times for 101 yards and three scores. Barrett Carter carried four times for 96 yards and a score, a 47-yarder for an early 7-0 lead.
Kayden McDonald's 9-yard TD run put North up two scores before Mountain View's Nathan Payne threw a 4-yard TD pass to Mason Kidd to trim the lead. Payne completed 13 of 28 passes for 230 yards a pair of TDs, and his top receivers were Mossiah Carter (four catches, 98 yards), Marcus Brand (two catches, 74 yards) and Zay Wilson (six catches, 51 yards).
North responded with Washington's 28-yard TD run late in the first quarter, then scored twice in the second quarter — a 49-yard TD pass from Washington to Seth Anderson and a 1-yard TD run by McFarlane.
Washington had a 3-yard TD run, Cody Leach had a 33-yard field goal and Washington tacked on a 15-yard TD run for a 52-7 lead in the third quarter.
Payne threw a 71-yard TD pass to Brand late in the third before McFarlane closed the scoring with a 49-yard TD run early in the fourth.


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El Hadjar complex: Importing crude steel to address supply issueSunday, 01 November 2020 16:13
ALGIERS- The Steel Complex of El-Hadjar in Annaba will be allowed to import crude steel in case of local production deficit, to solve the problem of supply of raw materials, said Minister of Industry, Ferhat Aït Ali Braham.
A laboratory for industrial innovation inaugurated in AlgiersThursday, 24 September 2020 15:45
ALGIERS- Minister of Industry Ferhat Ait Ali Braham on Thursday in Algiers inaugurated a laboratory for industrial innovation "Fablab."
Investment: Census of production assets in public, private sectorsSaturday, 15 August 2020 11:44
ALGIERS-Minister of Industry Ferhat Ali Braham on Thursday announced a census of the production assets in the public and private sectors in order to determine the added value brought to the economy.
Ferhat Ait Ali: Phosphate mega-project to be relaunched soon in east of AlgeriaThursday, 11 June 2020 09:53
ALGIERS - The integrated phosphate mining and processing mega-project in the east of the country will soon be relaunched after several months of shutdown, announced the Minister of Industry and Mining, Ferhat Ait Ali Braham.
Coronavirus: Exceptional facilities to import necessary raw materialsThursday, 02 April 2020 14:30
ALGIERS- Minister of Industry and Mining, Ferhat Aït Ali Braham announced, on Wednesday, exceptional customs and banking measures for importing needed raw materials to prevent the spread of coronavirus crisis.
Coronavirus: Ferhat Ait Ali orders public industrial groups to up productionMonday, 30 March 2020 20:38
ALGIERS- Minister of Industry and Mines Ferhat Ait Ali Braham ordered state-owned industrial groups to increase their production to meet the growing demand, as part of the actions to deal with coronavirus pandemic, the ministry said Monday in a statement.
Industry: Towards review of investment lawWednesday, 19 February 2020 16:57
ALGIERS – The law governing the industry sector will be reviewed in order to promote investments, profitability and productivity gain, said Tuesday Minister of Industry and Mines Ferhat Ait Ali Braham.
Car assembly: new specification note to be issued within two monthsSunday, 16 February 2020 20:38
ALGIERS- Minister of Industry and Mines Ferhat Aït Ali Braham, announced Sunday in Algiers that the new specifications on the automobile assembly activity will be promulgated within two months.
Industry minister calls for revision of investment lawsWednesday, 12 February 2020 16:35
ALGIERS – Minister of Industry and Mine Ferhat Ait Ali Brahim on Tuesday in Algiers called for the review of investment laws in a bid to attract more Algerian and foreign investors.


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Rockmont Blog
Behind the Scenes: How We Develop Rockmont's Program in Pursuit of Five Critical Objectives for our Campers
Part One: Self Confidence Parents tell us that in the days after their son has been to (...)
The Case for Camping
I believe in the power of a night spent camping in the woods. At Rockmont, we believe in (...)
Rounding Out Rockmont's Roster
Last week was opening week for Major League Baseball, and that means summer is right around the (...)
Father/Son Weekend 2018 – September Rafting Photos
Enjoy scrolling through the album below to see all the fun and adventure from our Rafting Trip at (...)
Next Door and a Million Miles Away
My wife and I and our three boys live in the Rock House at the geographic center of Camp (...)
Come and See
Sunday was a big, good day. And the camp is full now! Classic 3 campers have joined Summit (...)
Cross Country at Camp Rockmont
This summer Rockmont will be offering specialized Cross Country training for older campers. Led by (...)
All at Once
A story of thanks for God's outpouring of gifts. I married my wife, Sadie, on Saturday (...)
Grace Goes First
Gaining insight on blessings and affirmations from older cultures. When does a boy become a man? (...)


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A week course that teaches sustainable horticulture, urban greening and community building, to provide citizens with the skills necessary to increase community-supported urban green spaces. Looking for opportunities to volunteer? Beautiful RVA offers plenty of ways to get your hands dirty throughout the Richmond region.
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Cassius Conrad
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If this is indeed the new Osmo Pocket 2, how many of you will be upgrading? Loving the Osmo Pocket but I'm pretty sure that DJI has something up their sleeves for this one.
DJI - Capture Magic at hand
DJI - Capture Magic at hand


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We should stipulate at this point that neither of us have any idea how good this draft is going to be because the college season is barely underway. It sure looks nice, what with all those freshmen stacked up together in the lottery.
Best Companies rank: 11
《拉里·威尔莫夜间秀》(The Nightly Show with Larry Wilmore),Comedy Central,1月19日播出。威尔莫的主题喜剧秀将取代《科尔伯特报告》(The Colbert Report)在夜间11:30播出,这是深夜节目多样化的最新尝试。完全没有压力!
不错,2014年完全是一场灾难,只等着引爆。在《年内股市崩盘的概率有多大?》(Doomsday poll: 87% risk of stock crash by year-end)一文中,我们分析了今年年初以来的10大崩盘征兆。之后,我们又不断发现了更多新的妖魔。它们有如嘀嗒作响的定时炸弹,来自国会、最高法院、性问题、碳排放、石油巨头、国家安全局(NSA)、国税局(IRS)、茶党式财政紧缩。接踵而至,让人无法招架。
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Xi'an Xianyang International Airport outshone all other airports in China, with a punctuality rate of 82.3%.
Canadian schools continue to lose ground. Only three remain in the ranking compared with five last year and all fall about six places. Among these, Rotman School of Management at the University of Toronto is the highest ranked at 65, while Smith School of Business at Queen's University is bottom of the table in 100th place.
But, despite the incessant warnings from security researchers, many people are still turning to weak, easily guessable passwords to protect their devices.
The night's biggest winners may have been hosts Ms. Fey and Ms. Poehler, whose second time hosting the Hollywood Foreign Press Association's Beverly Hills, Calif., ceremony was just as successful as last year's show (a six-year ratings high with 19.7 million viewers).
在《招魂》一举成功后, 埃德·沃伦和罗琳·沃伦夫妇又推出了其续集《招魂2》。 影片讲述了1977年发生在伦敦的一个真实故事——恩菲德招灵事件。
Unfortunately for May, the big drop in the pound since the Brexit vote means her salary has plunged in dollar terms.
With most of our favorite shows on or approaching the dreaded holiday hiatus, it's time to reflect on the women that made the fall 2011 TV season so great.
vi. & vt
Angela Merkel's salary as German chancellor is set at 218,400 euros a year ($242,000). The chancellor got a 2% pay rise at the beginning of March, and will get another 2% next February.
Against: To some extent it's divided American audiences, perhaps damaging its hopes of winning best film.
Alternative fuels: For the first time since diesel-powered cars from Europe started arriving here in the 1950s, old first-person accounts about the joys of driving a diesel fall off to near zero. They are replaced by new first-person accounts about the joys of driving cars with ridiculously high horsepower like the 580-hp Chevrolet Camaro ZL1 and Dodge's Challenger SRT Hellfire with 707 hp. Elsewhere: fuel-cell cars with their longer cruising range move center-stage, stealing the limelight from battery-powered cars.
According to the National Business Daily, the 16 listed banks plan to return 356.2 billion yuan to shareholders as cash dividends in 2015, a decrease of 8.3 billion yuan from the previous year.
Homing in on a brand that excels at making the kind of vehicle you're looking for is an efficient way to focus your shopping and find the best car for you. The 2016 U.S. News Best Vehicle Brand awards recognize the best brands in four categories: Cars, SUVs, Trucks and Luxury.
《速度与激情6》帮助其另外两位男演员在我们榜单中获得较高的排名:范·迪塞尔(Vin Diesel)和已故的保罗·沃克(Paul Walker)。迪塞尔以8.87亿美元位居我们榜单第四。《速度与激情6》在全球斩获7.89亿美元的票房。仅仅几个月之前,他出演的《星际传奇3》(Riddick)在各大影院上映,仅收入9,800万美元票房,不过,这也足以使迪塞尔的排名领先于他的搭档沃克。
Except your Watson, he thinks it's hot.
今年,我最喜欢的人物报道可能是Kiki Zhao对中国目前读者最多的诗人之一余秀华激动人心的描写。余秀华是一个了不起的人。她患有脑瘫,过去41年里大部分时间生活在农村,在一张矮桌子上写作。高中没毕业的她说自己"在能读它们(名著)之前,我就知道如何写作了"。现在,她受邀前往斯坦福大学等地,她不愿被比作艾米丽·迪金森(Emily Dickinson)。
This so-called "playground for millionaires" is now open to everyone. The wallet-friendly hotels, guesthouses and self-catering apartments make tours affordable. With more than 100 divine islands scattered on the Indian ocean, many activities await, such as sunbathing, fishing, diving, sailing, and going on boat tours. The Seychelles islands are blessed with a warm tropical climate all year round, so you can go there whenever you want.
For: Oldman's seemingly effortless embodiment of the wartime leader has won almost universal
Even if Asian countries introduced regulation similar to that of thriving ETF markets elsewhere in the world, there are other hurdles. Mr Montanari says, for example, that most products presented to Asian investors relate to equities in their domestic market so the ETFs are not being used to give investors local exposure to foreign markets.
Denmark, for instance, is 88 percent white Danish today — hardly a majority in jeopardy. But a generation ago, in 1980, it was 97 percent white. The anti-immigrant Danish People's Party is now the second-largest party in the Danish Parliament. In Germany, where the foreign-born population shot up by approximately 75 percent between 2011 and 2015, the anti-immigrant, populist Alternative for Germany party is now drawing record support.
专家:房价涨势陆续放缓 下半年仍需稳字当头
The report also found that nearly a quarter of people use the same password for every site they are signed up to.
The overwhelming weight of the coverage of the oil market by analysts and commentators concentrates on the supply side and the volumes being produced and exported from Saudi Arabia or elsewhere. Supply is important but costs matter more.
Rose Wang
Bra discussions: Lindsay Lohan discusses her lingerie needs with a sales assistant in the Naked store in SoHo, New York on Tuesday . 来!内衣分析一下:周二在
We will make our skies blue again.
Mr Karl added: "We would have likely had a record [year] even without El , but it pushed it way over the top."
During the same period, Braziland Russia slashed their holdings to USD 261.7 billion and USD 108.9 billion,respectively.
It had hit a record low of 48 in September, and was 49.9 in October.
Blake Griffin's high school appearance isn't that far off from how he looks today. Just add some hair and some size to his biceps, and you've got Blake Griffin today.
The University of Tokyo (22nd) and Kyoto University (35th) have maintained their positions as the leading universities in Asia.


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Get back to this page: https://access.nyc.gov/programs/home-energy-assistance-program-heap/
Money for heat and utility expenses
Home Energy Assistance Program (HEAP) | NYC Human Resources Administration
1. How it Works
HEAP helps eligible households meet their home energy needs in the winter season. The money can pay for fuel, your utility source, equipment and/or repairs. The City pays the costs either directly to the company (heating benefit and/or utility benefit) or to the household (utility benefit only).
- The HEAP benefit opens November 2, 2020. The deadline to submit applications is March 15, 2021.
- The Emergency HEAP benefit opens January 4, 2021 and closes March 15, 2021 or until funds run out.
- You may still be able to receive benefits even if your utilities are included in your rent.
- Emergency benefits are available if your heat has been turned off or is in danger of being turned off.
- Requires proof of address, social security number, income, and your utility bills (if you pay utilities).
Next section:
RelatedCash & Expenses Everyone
More ways to get help with this program
Visit the website
Visit the HEAP website for more information.
Call 311
Ask for assistance with the Home Energy Assistance Program.
Call HEAP
Call the HEAP Infoline at 800-692-0557 if you have questions about HEAP or 212-331-3126 for operator assistance.
Call the Heat Line at 212-331-3150 if you have trouble paying your heat bill or are home-bound and need assistance with HEAP.
Visit a HEAP Office
Visit a HEAP office to get help in person.
2. Determine your eligibility
To be eligible, you should be able to answer yes to these questions:
- Do you have a child under six years old, an adult over 60 years old, or anyone with a permanent disability living with you?
- Are you and the people you live with US citizens or legal immigrants?
- Do you meet one of the following income guidelines?
- You receive SNAP benefits, Temporary Assistance, or Code A Supplemental Security Income.
- Your family is at or under the following income guidelines.
Family Size
Income in a month
For each additional person, add:
If you need emergency HEAP benefits, you should also be able to answer yes to these questions:
- Is your heat shut off or about to be shut off, or is your fuel tank a quarter full or less?
- Is the heating and/or electric bill in your name?
- Are your household's available resources:
- less than $3,000 if there is a member age 60 or older, or
- less than $2,000 if there is no member age 60 or older?
Take a short survey to see if you might be eligible for this or 30 other programs.Am I Eligible?
Next section:
3. What you need to include
It's important to include the right documents when you apply. Your documents show whether the program is right for you.
This page can help you choose the right documents for your application.
Proof of who you are
For each person applying, any one document:
- Driver's License
- Photo ID
- US Passport or Naturalization Certificate
- Adoption Papers, Hospital or Doctor's Records
- School Records
Or for each person applying, any two documents:
- Birth Certificate or Baptismal certificate
- Validated Social Security Card
- Statement from another person
Proof of where you live
For the household, any one document:
- Copy of most recent deed
- Current letter, lease, or rent receipt with home address from landlord
- Documentation from the New York City Housing Authority (NYCHA)
- Documentation from the New York City Human Resources Administration (HRA)
- Driver license issued within past six months
- Homeowners insurance policy
- ID card with address
- Lease signed by both landlord and tenant
- Property tax records or mortgage statements
- Statement from an employer or social services agency
- Statement from non-relative landlord
- Utility bill (gas, electric, or water)
Proof of your income
For each income source of each income earner, any one document:
Wages & Salary
- Letter from employer on company letterhead, signed and dated
- Paycheck stubs (from the last four weeks in a row that you worked)
- Business records
- Business records of earnings and expenses for the most recent three months in a row
Cash Assistance
- Cash Assistance budget letter
Income from Rent or Room and Board
- Check stub
- Ledger or rent receipt book
- Letter from roomer, boarder, or tenant
Military Pay
- Current award letter
- Current check stub
Child Support or Alimony
- Alimony check stub
- Child support check stub
- Letter from court
- Letter from person providing support
Unemployment Income
- Award letter or certificate
- Correspondence from NYS Department of Labor
- Bank statement showing the direct deposits
- Copy of the direct payment debit card
- Benefit statement
Social Security benefits (SSI, Dependent, Disability, Survivor's, or Retirement)
- Current award letter or certificate
- Current benefit check
Worker's Compensation
- Current award letter
- Current check stub
Veteran's Benefits
- Current award letter
- Current benefit check stub
Pensions, Annuities, or IRA
- Statement from pension or annuity
Interest, Dividends, or Royalties
- Statement from bank, credit union or financial institution
Support from Family Members
- Copies of canceled checks from family member
- Signed statement or letter from family member
Educational Grants or Loans
- Current award letter
- Current check stub
Proof of your expenses
For each expense, any one document:
Dependent Care Costs
- Court order
Shelter Expenses
- Current rent receipt
- Current lease
- Mortgage records
- Property tax records
- Sewer and water bills
- Fuel bills
- Utility bills
Proof of citizenship/immigration status
For each person applying, any one document:
- US birth certificate
- US passport
- US baptismal certificate
- Official US hospital or doctor records
- Green Card (copy of the front and back)
- Naturalization certificate
- Employment Authorization Card (I-688B or I-766)
- INS I-210 letter
- Memorandum of Creation of Record of Lawful Permanent Residence: INS form I-181
- INS form I-220B
- Arrival-Departure Record Card: INS form I-94
- Other INS documents, or correspondence to or from the INS, that shows that you are living in the US with the knowledge and permission of the INS
Proof of your resources
For each resource of each resource holder, any one document:
- Bank books
- Bank statement
- Burial agreement or burial insurance
- Copies of stocks, bonds, or securities
- Credit union records
- Deed or appraisal for real estate
- Sample check
Proof of your disability status
For each person with a disability, any one document:
- Statement from medical professional
- Proof of Social Security Disability (SSDI) or Supplemental Security Income (SSI)
Next section:
4. How to apply
Here are your options.
Apply by mail
Applications should be mailed to:
New York City Home Energy Assistance Program
P.O. Box 1401, Church Street Station
New York, NY, 10008
Apply by phone
To apply for Emergency HEAP you must first apply and be approved for regular HEAP. You can apply for Emergency HEAP over the phone by calling 212-331-3126.
Apply in person
- Find a HEAP office near you.
- Gather your personal documents and bring them with you to the office.
- You can print and fill out the application before you go in or complete it at the office.
Come back to it later.
We can send you a link to this page to help you get back to it when you're ready.
By providing your email address or phone number, you agree to be contacted by the City of New York regarding your ACCESS NYC public benefit screening results. Your activity on ACCESS NYC is anonymous, but providing your phone number may identify you and reveal that you used the website. To find out more about how the City might use the data you share with ACCESS NYC, visit the Terms of Use for NYC.gov, and Privacy Policy for NYC.gov.
More ways to get help with this program
Visit the website
Visit the HEAP website for more information.
Call 311
Ask for assistance with the Home Energy Assistance Program.
Call HEAP
Call the HEAP Infoline at 800-692-0557 if you have questions about HEAP or 212-331-3126 for operator assistance.
Call the Heat Line at 212-331-3150 if you have trouble paying your heat bill or are home-bound and need assistance with HEAP.
Visit a HEAP Office
Visit a HEAP office to get help in person.


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Wednesday 14th
Join the town for this year's Late Night Christmas Shopping
Free gifts for all children of Primary School age at Santa's Grotto
(Find Santa in NatWest Bank)
Complimentary Soup distributed by the Lions
Try one of the Lions famous Burgers or Hot Dogs!
Thursday 22nd
Lions Members and friends will wander around the town singing carols
Thursday 3rd
Club Business Meeting
The White Hart 8pm
Sunday 20th
Fun Day for the disabled with David Rowlands Fun Fair plus lots of other attractions
Attendance is by invitation only, if you know someone who might benefit from this event, or, you would like to offer to help in some way, please contact any Lions Member.
Memorial Hall and Manor Car Park (courtesy of Torridge DC)
Saturday 2nd
St Peter's Fair Week begins
A full week of entertainment and competions throughout the town.
Live Music in The Square
Wednesday 6th
Lions Junior Town Crier Competition
Entry forms at schools and groups
The Stage in The Square 6.30pm
followed by Live Music
Saturday 9th
Lions famous Bar-B-Q
Live Music (FREE!) in the Square
Thursday 4th
Club Business Meeting
The White Hart 8pm
Saturday 9th
Annual Charity Speedpool
proceeds split between MacMillan Nurses and Lions local Charities
Saturday 23rd
Annual Easter Egg Draw
proceeds to Lions local Charities
Friday 29th
Royal Wedding Celebrations
Internationally acclaimed show 'Bjorn Again' on the stage in the Manor Car Park
Wednesday 22nd
Holsworthy Fun Day
at Holsworthy Community College
Saturday 21st
'Bucket Shake' at Morrisons, Bude
a collection made by Lions Club Members
Over £500 collected for Devon & Cornwall Air Ambulances!
Thanks to all who supported this collection
Sunday 29th
Members Ted & Ted (Flaherty and England) are cycling in Ireland
donations for our "Winter Fun Day for the Disabled" to Lions Club Members
St Peter's Fair Week
visit www.stpetersfair.org.uk for full details
Wednesday 7th
Junior Town Crier Competition
Two separate competitions for different age groups
ask at school for details or phone a Lions member (numbers opposite)
Click here for photos of previous competitions
Saturday 10th
Pram & Wheelbarrow Race
5.30pm registration at the Scout Hut, Well Park car park
Click here for Rules and Guidance
Bar-B-Q in the Square
The last night of Fair, why not join the hundreds of revelers in the Square for
the fantastic 'Tribute Show', live on the stage, and enjoy a burger or hotdog from
the renowned Lions Barbeque
Saturday 31st
Great Tamar Duck Race
Win wonderfull prizes whilst helping our charities
Tickets available in the Square every Wednesday and weekends from June
(as well as from members of course!)
At Bridgrule Revel
Annual Duck Race 2010
Click here for Revel details and Bridgrule website
Thursday Oct 14th
Click here for more information ABOUT SPECTACLE DONATIONS
Sunday Nov 21st
Holsworthy Lions Winter Funday for the Disabled see gallery for previous years
Contact Lion Secretary John Cottle 01409 254 826 for details of registration
From the front page of the North Devon Journal Dec 2nd 2010
DRIVE TIME: Holsworthy Lions President Brian Withall helps out on the Bumping Cars. Picture: Ian Snell Ref: BNIS20101121A-001_C
HOLSWORTHY Lions fun day saw 480 disabled people from across the South West have a great time – and enjoy almost 1,000 free hot dogs and burgers between them.
The annual party with a disco inside the Memorial Hall and fair ground rides, a coconut shy, plate smashing and other fun activities outside, is a favourite for those with physical and mental disabilities and their carers.
Lions President Brian Withall said: "We have been running it for 20 years and they come from all over Devon and Cornwall and as far north as Weston-Super-Mare, and some come every year.
"It's a really good day, the fairground rides are left in place by Rowlands after the carnival and everything is free, the rides, the disco, the food, this year we got through a record 500 burgers, and more than 400 hot dogs and loads of candy floss."
Sunday Dec 5th
Holsworthy Lions erect the Christmas Trees in The Square
Anyone who would like to help the Lions deck the trees is welcome to come along
on Sunday morning at 10.30am
Click here for pictures of a previous year
Wednesday Dec 8th
Holsworthy Chamber of Trade Christmas Late Night Shopping
Holsworthy Lions Santa's Grotto with gifts for all children of primary school age
and of course Holsworthy Lions Bar-B-Que
Wednesday Dec 22nd
Lions Coffee Morning at the Memorial Hall
Holsworthy Lions Christmas 'Super Market Grab' winning tickets will be drawn at approximately 11.45am during this event
Lions Carol Singing
Holsworthy Lions will be singing at various locations throughout the town at various times in the run up to Christmas. A collection will be made in aid of the Holsworthy Lions Christmas activities, please give generously!
Wednesday Jan 12th
Lions Coffee Morning at the Memorial Hall
Holsworthy Lions Christmas 'Super Market Gra
Lions Senior Citizens Christmas Dinner at the Memorial Hall
Deatils from Lions Members. Date to be decided


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The price of Bitcoin went up 4.11 per cent of Wednesday, to trade at $12,416 and rising—a 2020 high—after digital payments provider PayPal announced it would allow customers to buy, sell and hold bitcoin and other virtual coins via its online wallets.
By early 2021, PayPal customers will be able to use cryptocurrencies to shop with its 26 million merchants on its network, the company said in a statement.
PayPal hopes the service will encourage global use of virtual coins and prepare its network for new digital currencies that may be developed by central banks and corporations, President and Chief Executive Dan Schulman told Reuters in an interview.
"We are working with central banks and thinking of all forms of digital currencies and how PayPal can play a role," he said.
The ability to buy, sell and hold cryptocurrencies will be rolled out to US users over the coming weeks, with plans to expand to Venmo (a mobile payment service owned by PayPal) and other countries by early 2021, the company said.
PayPal has 346 million active accounts around the world and processed $222 billion in payments in the second quarter of 2020.
The future of cryptocurrencies in India remains uncertain, with reports that the cabinet is to discuss a bill banning cryptocurrencies. This, after the Supreme Court overturned the RBI's own ban of crypto transactions that had been enacted in 2018.


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Penis extenders use penile traction to pull throughout the penis length. This causes the development of some small ruptures throughout this length which recover gradually resulting in tissue growth in the stretched area. And after a year or more, you'll observe the improvement in the length and girth of your member. Well, listed below are the top penis extenders of 2020:
The Quick Extender Pro
Well, if you're trying to find the very best penis extender of 2020, then you'll never ever go wrong with the Quick Extender Pro. It's perfect for enlarging your penis and increasing its curvature. This device uses a Double Strap Support (DSS) system where it's the only penis extender utilizing it giving you maximum stress to your penis or the very best comfort while maximizing its gains. Besides, its style allows you to expand your penis much faster and more dependably. Pro Extender Penis Extender Reviews
The device style was made by a New York-based organization called Innovatech design that has actually remained in the industry for a long time to comprehend the various customer requirements. Besides, the item has also gone through numerous modifications in its style to ensure the designers discover the ideal sweet spot that guarantees it provides the required stress and convenience for long-lasting results. Finally, you'll love to hear that it's an American and not a European brand. And though this does not guarantee much better efficiency, you will not experience such problems with consumer assistance.
Top Penis Stretchers On The Market
It comes with a special variation or curved for those with curved penis issues and will take only about six months or to start revealing outcomes for its users. And when you compare it with other extenders in the market, you will recognize that it produces higher tension and produces quicker outcomes. Overall, I would advise it to any user who wishes to increase the size of his penis. The Deluxe basic edition is the most perfect for users who focus on quality over spending plan while those low budget users can go for the value edition. Ensure you acquire this item from its main site to avoid getting scammed with inexpensive and phony products.
Pro Extender Penis Extender Reviews
Overall, the Quick Extender Pro provides you quality, is safe and effective. It is likewise pain-free and will assist you to remedy the curvature that results from Peyronie's disease. We found it for the lowest price here.
Easy to assemble and tidy
Easy to use
It's really comfy
It includes a 6 months money-back guarantee
It's not perfect for novice users as it produces much traction force
Phallosan Forte
Phallosan Strength is the 2nd top-rated penis extender in our review and this is primarily due to its proven effect in aligning and lengthening your penis. And besides, it is not agonizing, is trusted, and enables you to achieve your penis dream length inconspicuously. This gadget is produced by Texana manufacturing Limited which is based in Malta. From the different tests carried out with this device, it works and will help you repair lots of physiological problems in your penis without the requirement to get other items.
Full Penis Traction Device Review
And though its main function is to help you increase the size of your penis, it will also help you handle impotence offering you a stable and dependable repair for the condition. And besides girth smart, it'll likewise assist you acquire more length in your penis while also dealing with other problems like retraction penis, Incurvate penis, and many more. Overall, it runs as a bodybuilding fitness center devices given that it will allow you to enhance your self-confidence, gain a straight posture, and increase your penis size even within the first week.
Users have an assurance of getting a penis development
It has actually been tested and confirmed to be medically safe
Uses you a good value for your cash
It is 100% working and delivers lead to 3 months
You will have to use the device for a longer time daily
SizeGenetics device also falls in our list of the top three penis extenders in the market. it's quite small and you are therefore advised to use it while your penis is drooping. And when utilizing it, the gadget will slowly extend the penis just like what happens when you are lifting weights to boost muscle growth. The penis tissues will be promoted to grow and stretch more therefore increasing the length and girth of your dick.
Pro Extender Penis Extender Reviews
And besides, it's simple to use and you require to adorn it 5-8 hours daily; however you can use it in 2-hour increments until you reach the limit of 8 hours required or the very best outcomes. If you enjoy an active social life or if you generally invest lots of hours working, then this gadget is ideal for you. The variety of benefits you will acquire is directly proportional to the amount of time you'll be wearing this device.
The device is constructed utilizing premium materials that are not hypoallergenic. And its extender system allows you to achieve optimum lengthening results in a short time. Besides, it has been proven to be safe and highly effective based upon the countless user reviews online.
Easy to assemble
Its highly safe and effective
Offered in various plans to fit various users
It's quite pricey compared to similar items in the market
Getting a reputable male enhancement item in the market nowadays is harder due to different brand names and items assuring the very best outcomes but failing to provide. But with these leading three penis extenders, you can bypass surgery and enjoy watching your member grow longer and wider.
Females appreciate a big penis and it's natural for them to yearn for a larger member that will increase their sexual fulfillment. Also, as a man, you will be attracted to a bigger and much better penis that will improve your confidence when going down with your partner. Due to different genetic reasons, you will discover some guys having a huge penis while others have a smaller member. Thankfully, if you're disappointed with the size of your penis, then its length can be modified considering that it does not contain a bone.
That way, whether you have a little or normal size dick, then you can utilize penis extenders to help it grow longer. However, the majority of the marketplace brands are phony and retail poor quality products which apart from not providing the required outcomes could result in adverse effects to the users.
Do you wish to have the ability to have a more powerful erection? Well, if you are reading this write-up then chances are that you do. There are points that you need to learn about in order to achieve the very best feasible outcomes.
One of the most essential thing that you require to recognize is that there is no single trick that will help you out of this issue. There are some extremely good ways that are made use of in order to aid you out of it. If you have the ability to apply the right methods after that you must have the ability to accomplish the very best feasible results.
The first thing that you should do is learn how your penis is performing. It is feasible to do this by observing just how it goes put up and also the length of time it considers you to be able to get it back erect once again.
As soon as you have actually observed the manner in which your penis is carrying out then you ought to start considering your diet. You can do this by attempting a few different points such as taking zinc tablets. Zinc tablets are understood to help with the manufacturing of testosterone which is among the major reasons why your penis can having such a strong erection.
If you intend to be able to have a stronger erection then you likewise need to see to it that your diet has the right amount of zinc in it. If you do not eat sufficient zinc then you may wind up losing blood in your penis which can create an erection to fall. If you do not have adequate blood then your erection will certainly not be as strong and you might suffer from scratchy skin.
One more technique that can assist you out of the trouble is to utilize penis exercises. You can try utilizing penis workouts that are available totally free online. You can find plenty of these exercises online today. Nonetheless, you will certainly need to ensure that you do not make use of the incorrect exercises because they may refrain anything for you.
The very best part concerning making use of penis exercises is that they will certainly make certain that your erections remain solid over a long period of time. This means that you do not need to worry about your erections diminishing in a brief quantity of time. The good idea concerning making use of these workouts is that they can help you obtain the outcomes that you desire as well as require. without you having to bother with your erection stopping working.
Bear in mind that erections are something that are constructed over time. If you are willing to strive then you can attain better erections as well as take pleasure in the advantages that you are looking for. If you are serious about becoming an extra effective as well as satisfied lover after that you must check into these techniques today.
These methods can help you achieve the more powerful erection that you intend to have. Not just will your erections be better but you will additionally be able to delight in a great sex life with your partner. This will certainly likewise indicate that you will be able to offer your companion as much enjoyment as you desire.
If you wish to have the ability to have a stronger erection you will certainly additionally require to find out to maintain it properly. Your penis can become weaker in time and also if you do not stop it from obtaining weaker after that it will certainly be more difficult to accomplish a more powerful erection. If you wish to be able to get the results that you want after that you will certainly need to know exactly how to maintain your penis solid.
You can use penis workouts to aid you do this. You can do this by discovering these workouts and then you will understand what to do to keep your penis strong and improve it over time.
If you are ready to find out about penis workouts after that you will certainly have the ability to attain the outcomes that you intend to have. This will certainly make sure that you are happy with the way that your penis is carrying out.
Extend your penis and also you will certainly have a great deal of even more confidence. You will certainly feel wonderful, you will look fantastic as well as you will certainly feel better about on your own.
Something that can make your penis stretch out is extending it also far in one instructions. This will actually extend the skin, which is what will trigger you to have a smaller penis. It will certainly also lead to less blood circulation to the location and this might create a lack of experience.
Stretching your penis will certainly likewise extend the tendons. When you do this you are creating space between the ligaments and also the tissues. This is what will cause them to expand, which will certainly provide you a larger penis.
One more reason you should stretch out your penis is that having a small penis can be unpleasant. Individuals might be most likely to enhance you on your bigger penis and also they could also discover you appealing. When you obtain compliments, your self-confidence boosts.
The more tendons you have actually extended, the extra versatile your penis will certainly become. This is wonderful because your penis will have the ability to extend without becoming painful or inflamed.
Your ligaments likewise help your penis to hang straight and also when you have ligaments that are stretched, they will come to be tighter. This will certainly enable your penis to be a lot more reliable when you are doing sexual relations.
The last way to extend your penis is by exercising it. This exercise is called Jelqing. You can perform this workout at home by following some basic instructions. You will certainly require a water bottle, some lube, a mirror, a clean cloth and also a towel.
Extending your penis will assist to boost its length as well as girth in addition to the length as well as girth of the shaft. When you carry out these stretches, you will likewise enhance blood circulation to the area as well as this will certainly lead to better sexual pleasure for you as well as your partner.
You will certainly additionally raise the blood circulation to your penis as a result of stretching out your penis. This helps to lower the quantity of blood loss that creates you to climax quicker.
One of the most vital part of this exercise is to ensure that you are holding the penis in the right placement. You require to make sure that you are bending it from all-time low to make sure that you can see the summary of it. You wish to ensure that your thumb as well as forefinger are touching your penis while you are curved at the knees.
See to it that you don't flex your knees all the way down. Simply ensure that you are flexing the tip of your penis to the level with your lower abdominal areas.
See to it that you are holding the penis for a number of seconds before you pull it back up. so that you can really feel the stretch from the base to the top.
When you arrive of the stretch, see to it you draw it back out. simply make certain that you do not allow it slip back down.
You will be able to discover that your penis will certainly remain to stretch as long as you keep doing this workout. See to it that you do this exercise at the very least 4 times a week.
You will be stunned at just how your penis will certainly react when you start including even more exercises to your regimen. The better workouts will certainly make your penis larger than before.
When you are doing your penis exercises, make sure that you use lubricating substance on the penis prior to and after the workout. Lubricant will certainly help you to avoid tearing and also inflammation.
In summary, you must attempt to stretch out your penis routinely so that it remains stretched and healthy and balanced. You will certainly likewise have the ability to increase blood flow as well as improve your general stamina.


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Postmates Unlimited: Get Anything Delivered — Including Access to Today's Hottest Artists
ICYMI: Postmates expanded its Unlimited subscription service this month to offer free delivery of anything, from anywhere. To celebrate, we're offering some pretty insane "Unlimited" experiences.
This weekend at the Day N Night Music Festival, Kendrick Lamar and Chance the Rapper performed killer sets at the Postmates Main Stage — and between acts, festivalgoers hung out in our Unlimited lounge where artists Kamaiyah and Rich Chigga ordered "unlimited" amounts of Chick-fil-A via Postmates for the crowd. #takeusback
Experiencing FOMO about missing the show? Have no fear, we're helping Galantis celebrate the launch of their latest EP with a secret show in San Francisco this Tuesday, September 12. The ONLY way you can unlock the show is to sign up for Postmates Unlimited.
When we say we aim to be the go-to for "anything delivered, anywhere" — we mean it, especially for music fans. From your favorite restaurants to drinks on-demand, to our Justin Bieber pop-up store to unreleased Katy Perry music, sign up for Postmates Unlimited and you'll never pay for delivery or miss a beat.


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Did you know Sanjay Dutt was scared to shake a leg with Madhuri Dixit in a song?
The entire country is currently praying for the quick recovery of Sanjay Dutt who has been diagnosed with lung cancer. Reports state that he is going to jet off to the US soon to undergo further treatment. The actor had been already admitted to the Lilavati Hospital in Mumbai a few days back. That was because of complaints of breathlessness. He was discharged soon after but announced on social media about taking a break owing to medical reasons. Meanwhile, this piece of news has now shocked everyone.
Sanjay Dutt is one of the most popular actors who has been an instrumental part of Bollywood for a long time. Today, we will be talking about a particular incident related to the actor's life. We all know that he has worked with Madhuri Dixit in numerous movies. The 1990 action drama titled Thanedaar also happens to be one of them. It is said that Sanjay Dutt was scared after getting to know that he is going to dance with Madhuri in the song 'Tamma Tamma Loge.'
The actor reportedly said that the actress was a dancing queen and that he is nothing in front of her. However, he took up the challenge later on and practiced for around 16 days to perfectly ace the steps. And as we know, the rest is history. The audience loved the duo's chemistry not only in the song but also in the movie. Meanwhile, Sanjay Dutt and Madhuri Dixit have collaborated after a long hiatus in the movie Kalank that released in 2019.
Check out the video below:
Also Read: Maanyata Dutt releases statement after Sanjay Dutt's lung cancer diagnosis; Says, 'We will emerge as winners'
Credits :Youtube
Anonymous 3 months ago
Madhuri Dixit-Best actress ever and forever no comparison at all with anyone
Anonymous 3 months ago
madhuri dixit -best actresses ever and forever no comparison with anyone at all
Anonymous 3 months ago
Madhuri is an incredible Dancer
Anonymous 3 months ago
But he was shaking her for five years
Anonymous 3 months ago
Just praying for Sanjay Dutt to recover completely and live happy and long.