Environmental Factors Shape Sediment Anammox Bacterial Communities in Hypernutrified Jiaozhou Bay,China

Bacterial anaerobic ammonium oxidation (anammox) is an important process in the marine nitrogen cycle. Because ongoing eutrophication of coastal bays contributes significantly to the formation of low-oxygen zones, monitoring of the anammox bacterial community offers a unique opportunity for assessment of anthropogenic perturbations in these environments. The current study used targeting of 16S rRNA and hzo genes to characterize the composition and structure of the anammox bacterial community in the sediments of the eutrophic Jiaozhou Bay, thereby unraveling their diversity, abundance, and distribution. Abundance and distribution of hzo genes revealed a greater taxonomic diversity in Jiaozhou Bay, including several novel clades of anammox bacteria. In contrast, the targeting of 16S rRNA genes verified the presence of only “Candidatus Scalindua,” albeit with a high microdiversity. The genus “Ca. Scalindua” comprised the apparent majority of active sediment anammox bacteria. Multivariate statistical analyses indicated a heterogeneous distribution of the anammox bacterial assemblages in Jiaozhou Bay. Of all environmental parameters investigated, sediment organic C/organic N (OrgC/OrgN), nitrite concentration, and sediment median grain size were found to impact the composition, structure, and distribution of the sediment anammox bacterial community. Analysis of Pearson correlations between environmental factors and abundance of 16S rRNA and hzo genes as determined by fluorescent real-time PCR suggests that the local nitrite concentration is the key regulator of the abundance of anammox bacteria in Jiaozhou Bay sediments.Anaerobic ammonium oxidation (anammox, NH₄⁺ + NO₂⁻ → N₂ + 2H₂O) was proposed as a missing N transformation pathway decades ago. It was found 20 years later to be mediated by bacteria in artificial environments, such as anaerobic wastewater processing systems (see reference 32 and references therein). Anammox in natural environments was found even more recently, mainly in O₂-limited environments such as marine sediments (28, 51, 54, 67, 69) and hypoxic or anoxic waters (10, 25, 39-42). Because anammox may remove as much as 30 to 70% of fixed N from the oceans (3, 9, 64), this process is potentially as important as denitrification for N loss and bioremediation (41, 42, 73). These findings have significantly changed our understanding of the budget of the marine and global N cycles as well as involved pathways and their evolution (24, 32, 35, 72). Studies indicate variable anammox contributions to local or regional N loss (41, 42, 73), probably due to distinct environmental conditions that may influence the composition, abundance, and distribution of the anammox bacteria. However, the interactions of anammox bacteria with their environment are still poorly understood.The chemolithoautotrophic anammox bacteria (64, 66) comprise the new Brocadiaceae family in the Planctomycetales, for which five Candidatus genera have been described (see references 32 and 37 and references therein): “Candidatus Kuenenia,” “Candidatus Brocadia,” “Candidatus Scalindua,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia.” Due to the difficulty of cultivation and isolation, anammox bacteria are not yet in pure culture. Molecular detection by using DNA probes or PCR primers targeting the anammox bacterial 16S rRNA genes has thus been the main approach for the detection of anammox bacteria and community analyses (58). However, these studies revealed unexpected target sequence diversity and led to the realization that due to biased coverage and specificity of most of the PCR primers (2, 8), the in situ diversity of anammox bacteria was likely missed. Thus, the use of additional marker genes for phylogenetic analysis was suggested in hopes of better capturing the diversity of this environmentally important group of bacteria. By analogy to molecular ecological studies of aerobic ammonia oxidizers, most recent studies have attempted to include anammox bacterium-specific functional genes. All anammox bacteria employ hydrazine oxidoreductase (HZO) (= [Hzo]₃) to oxidize hydrazine to N₂ as the main source for a useable reductant, which enables them to generate proton-motive force for energy production (32, 36, 65). Phylogenetic analyses of Hzo protein sequences revealed three sequence clusters, of which the cladistic structure of cluster 1 is in agreement with the anammox bacterial 16S rRNA gene phylogeny (57). The hzo genes have emerged as an alternative phylogenetic and functional marker for characterization of anammox bacterial communities (43, 44, 57), allowing the 16S rRNA gene-based investigation methods to be corroborated and improved.The contribution of anammox to the removal of fixed N is highly variable in estuarine and coastal sediments (50). For instance, anammox may be an important pathway for the removal of excess N (23) or nearly negligible (48, 54, 67, 68). This difference may be attributable to a difference in the structure and composition of anammox bacterial communities, in particular how the abundance of individual cohorts depends on particular environmental conditions. Anthropogenic disturbance with variable source and intensity of eutrophication and pollution may further complicate the anammox bacterium-environment relationship.Jiaozhou Bay is a large semienclosed water body of the temperate Yellow Sea in China. Eutrophication has become its most serious environmental problem, along with red tides (harmful algal blooms), species loss, and contamination with toxic chemicals and harmful microbes (14, 15, 21, 61, 71). Due to different sources of pollution and various levels of eutrophication across Jiaozhou Bay (mariculture, municipal and industrial wastewater, crude oil shipyard, etc.), a wide spectrum of environmental conditions may contribute to a widely varying community structure of anammox bacteria. This study used both 16S rRNA and hzo genes as targets to measure their abundance, diversity, and spatial distribution and assess the response of the resident anammox bacterial community to different environmental conditions. Environmental factors with potential for regulating the sediment anammox microbiota are discussed.     

Impact of Temperature on Ladderane Lipid Distribution in Anammox Bacteria

Anaerobic ammonium-oxidizing (anammox) bacteria have the unique ability to synthesize fatty acids containing linearly concatenated cyclobutane rings, termed “ladderane lipids.” In this study we investigated the effect of temperature on the ladderane lipid composition and distribution in anammox enrichment cultures, marine particulate organic matter, and surface sediments. Under controlled laboratory conditions we observed an increase in the amount of C₂₀ [5]-ladderane fatty acids compared with the amount of C₁₈ [5]-ladderane fatty acids with increasing temperature and also an increase in the amount of C₁₈ [5]-ladderane fatty acids compared with the amount of C₂₀ [5]-ladderane fatty acids with decreasing temperature. Combining these data with results from the natural environment showed a significant (R² = 0.85, P = <0.0001, n = 121) positive sigmoidal relationship between the amounts of C₁₈ and C₂₀ [5]-ladderane fatty acids and the in situ temperature; i.e., there is an increase in the relative abundance of C₁₈ [5]-ladderane fatty acids at lower temperatures and vice versa, particularly at temperatures between 12°C and 20°C. Novel shorter (C₁₆) and longer (C₂₂ to C₂₄) ladderane fatty acids were also identified, but their relative amounts were small and did not change with temperature. The adaptation of ladderane fatty acid chain length to temperature changes is similar to the regulation of common fatty acid composition in other bacteria and may be the result of maintaining constant membrane fluidity under different temperature regimens (homeoviscous adaptation). Our results can potentially be used to discriminate between the origins of ladderane lipids in marine sediments, i.e., to determine if ladderanes are produced in situ in relatively cold surface sediments or if they are fossil remnants originating from the warmer upper water column.Anaerobic ammonium-oxidizing (anammox) bacteria possess the unique ability to oxidize NH₄⁺ with NO₂⁻ to N₂ under anoxic conditions (42). Since the discovery of the anammox process in a wastewater treatment plant in the Netherlands (21), studies have indicated that anammox bacteria are omnipresent in low-oxygen environments around the world. Anammox therefore forms an important link in both the oceanic (4, 7, 17, 18, 31) and freshwater (14, 33) nitrogen cycles. Unlike other Planctomycetes, anammox bacteria contain a unique “organelle” called the anammoxosome (19, 37, 44-46). The membrane of this compartment contains unusual “ladderane” lipids (37). The core ladderane lipids consist of C₁₈ and C₂₀ fatty acids containing either 3 or 5 linearly concatenated cyclobutane rings, which are ester bound to a glycerol backbone or ether bound as alkyl chains (35). In addition, the intact polar lipids containing the core lipid structures may have different types of polar head groups, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylglycerol (PG) (1, 22). In silico density simulation modeling experiments with a ladderane lipid-containing membrane (glycerol-bound mixed ether-ester containing both ladderane moieties) have indicated that ladderane lipids could provide a denser cell membrane than conventional membrane lipids (37). Since the anammoxosome appears to be impenetrable to fluorophores, the ladderane membrane could function in cell energy conservation (37, 44).Experimental evidence has shown that anammox bacteria isolated from wastewater treatment reactors grow over a wide range of temperatures (20 to 43°C) and have an optimum temperature of about 35°C (39). In the natural environment the anammox process has been reported to occur at temperatures as low as −2.5°C in sea ice (5, 26) and as high as 70°C in hot springs and hydrothermal vent areas (3, 12). Furthermore, “Candidatus Scalindua spp.” has been successfully enriched from marine sediment (Gullmarsfjord, Sweden) in sequencing batch reactors at temperatures of 15 and 20°C (43). In other bacteria containing common fatty acids temperature adaptation can be achieved by (among other things) modifying the composition of the membrane bilayers to deal with alterations in membrane viscosity due to changes in temperature. This process has been well documented and is termed “homeoviscous adaptation”; i.e., the fatty acid composition is changed to maintain membrane fluidity (23, 27, 34, 40). Currently, it is not known how anammox bacteria, with their highly unusual ladderane lipids, react to temperature. To investigate this, we analyzed the ladderane lipid composition of anammox bacteria grown at different temperatures in sequencing batch reactors and in samples from different natural environments covering a wide range of temperatures.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Anaerobic Biodegradation of n-Hexadecane by a Nitrate-Reducing Consortium

Nitrate-reducing enrichments, amended with n-hexadecane, were established with petroleum-contaminated sediment from Onondaga Lake. Cultures were serially diluted to yield a sediment-free consortium. Clone libraries and denaturing gradient gel electrophoresis analysis of 16S rRNA gene community PCR products indicated the presence of uncultured alpha- and betaproteobacteria similar to those detected in contaminated, denitrifying environments. Cultures were incubated with H₃₄-hexadecane, fully deuterated hexadecane (d₃₄-hexadecane), or H₃₄-hexadecane and NaH¹³CO₃. Gas chromatography-mass spectrometry analysis of silylated metabolites resulted in the identification of [H₂₉]pentadecanoic acid, [H₂₅]tridecanoic acid, [1-¹³C]pentadecanoic acid, [3-¹³C]heptadecanoic acid, [3-¹³C]10-methylheptadecanoic acid, and d₂₇-pentadecanoic, d₂₅-, and d₂₄-tridecanoic acids. The identification of these metabolites suggests a carbon addition at the C-3 position of hexadecane, with subsequent β-oxidation and transformation reactions (chain elongation and C-10 methylation) that predominantly produce fatty acids with odd numbers of carbons. Mineralization of [1-¹⁴C]hexadecane was demonstrated based on the recovery of ¹⁴CO₂ in active cultures.Linear alkanes account for a large component of crude and refined petroleum products and, therefore, are of environmental significance with respect to their fate and transport (38). The aerobic activation of alkanes is well documented and involves monooxygenase and dioxygenase enzymes in which not only is oxygen required as an electron acceptor but it also serves as a reactant in hydroxylation (2, 16, 17, 32, 34). Alkanes are also degraded under anoxic conditions via novel degradation strategies (34). To date, there are two known pathways of anaerobic n-alkane degradation: (i) alkane addition to fumarate, commonly referred to as fumarate addition, and (ii) a putative pathway, proposed by So et al. (25), involving carboxylation of the alkane. Fumarate addition proceeds via terminal or subterminal addition (C-2 position) of the alkane to the double bond of fumarate, resulting in the formation of an alkylsuccinate. The alkylsuccinate is further degraded via carbon skeleton rearrangement and β-oxidation (4, 6, 8, 12, 13, 21, 37). Alkane addition to fumarate has been documented for a denitrifying isolate (21, 37), sulfate-reducing consortia (4, 8, 12, 13), and five sulfate-reducing isolates (4, 6-8, 12). In addition to being demonstrated in these studies, fumarate addition in a sulfate-reducing enrichment growing on the alicyclic alkane 2-ethylcyclopentane has also been demonstrated (23). In contrast to fumarate addition, which has been shown for both sulfate-reducers and denitrifiers, the putative carboxylation of n-alkanes has been proposed only for the sulfate-reducing isolate strain Hxd3 (25) and for a sulfate-reducing consortium (4). Experiments using NaH¹³CO₃ demonstrated that bicarbonate serves as the source of inorganic carbon for the putative carboxylation reaction (25). Subterminal carboxylation of the alkane at the C-3 position is followed by elimination of the two terminal carbons, to yield a fatty acid that is one carbon shorter than the parent alkane (4, 25). The fatty acids are subject to β-oxidation, chain elongation, and/or C-10 methylation (25).In this study, we characterized an alkane-degrading, nitrate-reducing consortium and surveyed the metabolites of the consortium incubated with either unlabeled or labeled hexadecane in order to elucidate the pathway of n-alkane degradation. We present evidence of a pathway analogous to the proposed carboxylation pathway under nitrate-reducing conditions.     

mcrA-Targeted Real-Time Quantitative PCR Method To Examine Methanogen Communities

Methanogens are of great importance in carbon cycling and alternative energy production, but quantitation with culture-based methods is time-consuming and biased against methanogen groups that are difficult to cultivate in a laboratory. For these reasons, methanogens are typically studied through culture-independent molecular techniques. We developed a SYBR green I quantitative PCR (qPCR) assay to quantify total numbers of methyl coenzyme M reductase α-subunit (mcrA) genes. TaqMan probes were also designed to target nine different phylogenetic groups of methanogens in qPCR assays. Total mcrA and mcrA levels of different methanogen phylogenetic groups were determined from six samples: four samples from anaerobic digesters used to treat either primarily cow or pig manure and two aliquots from an acidic peat sample stored at 4°C or 20°C. Only members of the Methanosaetaceae, Methanosarcina, Methanobacteriaceae, and Methanocorpusculaceae and Fen cluster were detected in the environmental samples. The three samples obtained from cow manure digesters were dominated by members of the genus Methanosarcina, whereas the sample from the pig manure digester contained detectable levels of only members of the Methanobacteriaceae. The acidic peat samples were dominated by both Methanosarcina spp. and members of the Fen cluster. In two of the manure digester samples only one methanogen group was detected, but in both of the acidic peat samples and two of the manure digester samples, multiple methanogen groups were detected. The TaqMan qPCR assays were successfully able to determine the environmental abundance of different phylogenetic groups of methanogens, including several groups with few or no cultivated members.Methanogens are integral to carbon cycling, catalyzing the production of methane and carbon dioxide, both potent greenhouse gases, during organic matter degradation in anaerobic soils and sediment (8). Methanogens are widespread in anaerobic environments, including tundra (36), freshwater lake and wetland sediments (9, 12), estuarine and marine sediments (2), acidic peatlands (4, 14), rice field soil (10, 16), animal guts (41), landfills (30), and anaerobic digesters treating animal manure (1), food processing wastewater (27), and municipal wastewater and solid waste (37, 57). Methane produced in anaerobic digesters may be captured and used for energy production, thus offsetting some or all of the cost of operation and reducing the global warming potential of methane release to the atmosphere.Methanogens are difficult to study through culture-based methods, and therefore many researchers have instead used culture-independent techniques to study methanogen populations. The 16S rRNA gene is the most widely used target for gene surveys, and a number of primers and probes have been developed to target methanogen groups (9, 11, 31, 36, 38, 40, 46, 48, 57). To eliminate potential problems with nonspecific amplification, some researchers have developed primers for the gene sequence of the α-subunit of the methyl coenzyme M reductase (mcrA) (17, 30, 49). The Mcr is exclusive to the methanogens with the exception of the methane-oxidizing Archaea (18) and shows mostly congruent phylogeny to the 16S rRNA gene, allowing mcrA analysis to be used in conjunction with, or independently of, that of the 16S rRNA gene (3, 30, 49). A number of researchers have examined methanogen communities with mcrA and have found uncultured clades quite different in sequence from cultured methanogen representatives (9, 10, 12, 14, 17, 22, 28, 47).Previous studies described methanogen communities by quantitation of different clades through the use of rRNA-targeted or rRNA gene-targeted probes with techniques such as dot blot hybridization (1, 27, 37, 38, 48) and fluorescent in situ hybridization (11, 40, 44, 57). Real-time quantitative PCR (qPCR) is an alternate technique capable of determining the copy number of a particular gene present in the DNA extracted from an environmental sample. Only a few studies have used qPCR to quantitatively examine different clades within methanogen communities, and most of these studies have exclusively targeted the 16S rRNA gene (19, 41, 42, 54-56). Far fewer researchers have used qPCR to quantify methanogen clades by targeting the mcrA (21, 34, 45), and these studies were limited to only a few phylogenetic groups.In this paper we present a methodology for determining methanogen gene copy numbers through the use of qPCR targeting the mcrA. Methanogens were quantified in total using methanogen-specific primers in SYBR green assays and also as members of nine different phylogenetic groups using TaqMan probes targeting specific subsets of methanogens.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Siderophores Are Not Involved in Fe(III) Solubilization during Anaerobic Fe(III) Respiration by Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 respires a wide range of anaerobic electron acceptors, including sparingly soluble Fe(III) oxides. In the present study, S. oneidensis was found to produce Fe(III)-solubilizing organic ligands during anaerobic Fe(III) oxide respiration, a respiratory strategy postulated to destabilize Fe(III) and produce more readily reducible soluble organic Fe(III). In-frame gene deletion mutagenesis, siderophore detection assays, and voltammetric techniques were combined to determine (i) if the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration were synthesized via siderophore biosynthesis systems and (ii) if the Fe(III)-siderophore reductase was required for respiration of soluble organic Fe(III) as an anaerobic electron acceptor. Genes predicted to encode the siderophore (hydroxamate) biosynthesis system (SO3030 to SO3032), the Fe(III)-hydroxamate receptor (SO3033), and the Fe(III)-hydroxamate reductase (SO3034) were identified in the S. oneidensis genome, and corresponding in-frame gene deletion mutants were constructed. ΔSO3031 was unable to synthesize siderophores or produce soluble organic Fe(III) during aerobic respiration yet retained the ability to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. ΔSO3034 retained the ability to synthesize siderophores during aerobic respiration and to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. These findings indicate that the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration are not synthesized via the hydroxamate biosynthesis system and that the Fe(III)-hydroxamate reductase is not essential for respiration of Fe(III)-citrate or Fe(III)-nitrilotriacetic acid (NTA) as an anaerobic electron acceptor.Bacterial electron transfer to sparingly soluble electron acceptors is a critical component of a wide variety of environmental and energy-generating processes, including biogeochemical cycling of metals, degradation of natural and contaminant organic matter, weathering of clays and minerals, biomineralization of Fe-bearing minerals, reductive precipitation of toxic metals and radionuclides, and electricity generation in microbial fuel cells (17, 33, 34). Anaerobic and facultatively anaerobic bacteria capable of respiring sparingly soluble (<10⁻²⁵ M at pH 7) Fe(III) oxides are ubiquitous in nature and may be found in marine, freshwater, and terrestrial environments, including metal- and radionuclide-contaminated subsurface aquifers (25, 34). Fe(III)-respiring prokaryotes are also deeply rooted and scattered throughout the domains Bacteria and Archaea (possibly indicating an ancient metabolic process) and include hyperthermophiles, psychrophiles, acidophiles, and extreme barophiles (34). Despite their potential environmental, energy-generating, and evolutionary significance, the molecular details of microbial Fe(III) respiration remain unclear.Fe(III)-respiring, neutrophilic bacteria are presented with a unique physiological challenge: they are required to respire anaerobically on electron acceptors found largely as sparingly soluble Fe(III) oxides presumably unable to contact periplasm- or inner membrane (IM)-localized electron transport systems. To overcome this problem, Fe(III)-respiring bacteria are postulated to employ novel respiratory strategies not found in other bacteria (e.g., aerobes, denitrifiers, sulfate-reducing bacteria, and methanogens) that respire soluble electron acceptors (17, 38). The novel respiratory strategies include (i) a direct-contact pathway in which terminal Fe(III) reductases are secreted to the cell outer membrane (OM), where they contact and deliver electrons directly to external Fe(III) oxides (18, 23, 40, 42, 48, 57, 64, 67), (ii) a two-step electron shuttling pathway in which bacterially reduced endogenous or exogenous electron shuttles deliver electrons to external Fe(III) oxides in a second (abiotic) electron transfer reaction (11, 26, 39, 45), and (iii) a two-step Fe(III) chelation (solubilization) pathway in which Fe(III) oxides are first nonreductively dissolved by endogenously synthesized organic ligands prior to reduction of the resulting soluble organic Fe(III) [Fe(III) bound to an organic molecule] complexes (36, 59).Candidate organic ligands for production of soluble organic Fe(III) during anaerobic Fe(III) oxide respiration include siderophores, the Fe(III)-chelating compounds synthesized and secreted by a wide variety of bacteria and fungi for solubilization and subsequent assimilation of otherwise inaccessible Fe(III) substrates (12, 44, 49, 63). Hydroxamate-type siderophores are produced via N⁶ hydroxylation and N⁶ acylation of l-ornithine and, in some cases, cyclization to macrocyclic ring structures (13). The macrocyclic siderophores bisucaberin and putrebactin, for example, are two structural analogs of the cyclic bis(hydroxamate) siderophore alcaligin, synthesized by Aliivibrio salmonicida and Shewanella putrefaciens strain 200, respectively (27, 32, 65). After transport across the cell envelope via a TonB-dependent pathway, Fe(III) is subsequently released from the Fe(III)-siderophore complex by ligand exchange reactions promoted by siderophore ligand hydrolysis and/or protonation or by Fe(III)-siderophore reduction and release of Fe(II) to acceptor ligands (9, 66).The main objectives of the present study were to determine (i) if the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration are synthesized by Fe(III)-siderophore biosynthesis systems and (ii) if Fe(III)-siderophore reductases are required for respiration of soluble organic Fe(III) as an anaerobic electron acceptor. The experimental strategy for this study included (i) identification of genes encoding the siderophore biosynthesis and Fe(III)-siderophore reductase systems in the S. oneidensis genome, (ii) generation of in-frame deletions in the corresponding siderophore biosynthesis and Fe(III)-siderophore reductase genes, (iii) tests of the resulting siderophore biosynthesis mutants for production of siderophores and soluble organic Fe(III) during aerobic and anaerobic Fe(III) oxide respiration, and (iv) tests of the resulting Fe(III)-siderophore reductase mutants for respiration of soluble organic Fe(III) as an anaerobic electron acceptor.     

Identification and Characterization of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase from Bacillus megaterium

A gene that codes for a novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase, designated PhaZ1, has been identified in the genome of Bacillus megaterium. A native PHB (nPHB) granule-binding assay showed that purified soluble PhaZ1 had strong affinity for nPHB granules. Turbidimetric analyses revealed that PhaZ1 could rapidly degrade nPHB granules in vitro without the need for protease pretreatment of the granules to remove surface proteins. Notably, almost all the final hydrolytic products produced from the in vitro degradation of nPHB granules by PhaZ1 were 3-hydroxybutyric acid (3HB) monomers. Unexpectedly, PhaZ1 could also hydrolyze denatured semicrystalline PHB, with the generation of 3HB monomers. The disruption of the phaZ1 gene significantly affected intracellular PHB mobilization during the PHB-degrading stage in B. megaterium, as demonstrated by transmission electron microscopy and the measurement of the PHB content. These results indicate that PhaZ1 is functional in intracellular PHB mobilization in vivo. Some of these features, which are in striking contrast with those of other known nPHB granule-degrading PhaZs, may provide an advantage for B. megaterium PhaZ1 in fermentative production of the biotechnologically valuable chiral compound (R)-3HB.Polyhydroxyalkanoates (PHAs) are a group of polyesters that are produced by numerous bacteria as carbon and energy storage materials in response to nutritional stress (13, 27, 29). Poly(3-hydroxybutyrate) (PHB) is the most common and intensively studied PHA. Intracellular native PHB (nPHB) granules are composed of a hydrophobic PHB core and a surface layer consisting of proteins and phospholipids (13). The PHB of intracellular nPHB granules is in an amorphous state. When intracellular nPHB granules are exposed to extracellular environments due to cell death and lysis, the amorphous PHB is transformed into a denatured semicrystalline state. nPHB granules subjected to physical damage or solvent extraction to remove the surface layer can also crystallize into denatured PHB (dPHB) (13, 15). Artificial PHB (aPHB) granules, in which PHB is in an amorphous state, can be prepared from semicrystalline dPHB and detergents (1, 11, 23, 31).Various extracellular PHB depolymerases (PhaZs) that are secreted by many PHB-degrading bacteria have been demonstrated to specifically degrade dPHB (13, 14, 37). One exception is that PhaZ7, an extracellular PHB depolymerase secreted by Paucimonas lemoignei, displays unusual substrate specificity for amorphous PHB, with 3-hydroxybutyrate (3HB) oligomers as the main products of enzymatic hydrolysis (7). PhaZ7 exhibits no enzymatic activity toward dPHB. So far, a growing number of intracellular PHB depolymerases have been characterized. The intracellular PHB depolymerase PhaZa1 of Ralstonia eutropha (also called Cupriavidus necator) H16 has recently been established to be especially important for the intracellular mobilization of accumulated PHB (42). The main in vitro hydrolytic products of PhaZa1 degradation of amorphous aPHB are 3HB oligomers (31). PhaZd1, another intracellular PHB depolymerase of R. eutropha H16, shows no significant amino acid similarity to PhaZa1. The in vitro hydrolytic products of PhaZd1 degradation of amorphous aPHB are also 3HB oligomers. A 3HB monomer is rarely detected as a hydrolytic product (1). The intracellular PHB depolymerase PhaZ of Paracoccus denitrificans was reported previously to degrade protease-treated nPHB granules in vitro, with the release of 3HB dimers and oligomers as the main hydrolytic products (6). Recently, we have identified a novel intracellular PHB depolymerase from Bacillus thuringiensis serovar “israelensis” (39). The B. thuringiensis PhaZ shows no significant amino acid similarity to any known PHB depolymerase. This PhaZ has strong amorphous PHB-hydrolyzing activity and can release a considerable amount of 3HB monomers by the hydrolysis of trypsin-treated nPHB granules (39). It is of note that purified PhaZd1 from R. eutropha, PhaZ from P. denitrificans, and PhaZ from B. thuringiensis need pretreatment of nPHB granules with protease to remove surface proteins for PHB degradation (1, 6, 39). They show only very little or no activity toward nPHB granules without trypsin pretreatment. It has been demonstrated previously that these intracellular PHB depolymerases cannot hydrolyze dPHB (1, 31, 39).(R)-3HB, a biotechnologically valuable chiral compound, has been widely used for syntheses of antibiotics, vitamins, and pheromones (3, 30, 38). One way to produce (R)-3HB is heterologous coexpression of a PHB synthetic operon and a gene encoding an amorphous PHB-degrading PhaZ in Escherichia coli (3, 18, 25, 33, 38). A common problem encountered by this method is that oligomeric and dimeric forms of 3HB often constitute a major portion of the products of enzymatic hydrolysis, thus requiring further hydrolysis by 3HB oligomer hydrolase or heating under alkaline conditions to generate 3HB monomers (3, 18, 25, 33).Bacillus megaterium genes involved in the biosynthesis of nPHB granules have been cloned from strain ATCC 11561 and characterized previously (19, 21, 22). A gene encoding the extracellular PHB depolymerase PhaZ from B. megaterium was recently cloned from strain N-18-25-9 (34). However, little is known about B. megaterium genes involved in the intracellular mobilization of PHB. In this study, we have identified in B. megaterium ATCC 11561 an intracellular PHB depolymerase that could rapidly degrade nPHB granules in vitro without the need for trypsin pretreatment of the nPHB granules. Moreover, almost all the in vitro hydrolytic products released from the degradation of amorphous PHB by this PhaZ were 3HB monomers. This PhaZ could also hydrolyze dPHB with the generation of 3HB monomers. Thus, it appears to be a novel intracellular PHB depolymerase and may have promising potential for biotechnological application in the production of enantiomerically pure (R)-3HB monomers.     

Substrate-Level Phosphorylation Is the Primary Source of Energy Conservation during Anaerobic Respiration of Shewanella oneidensis Strain MR-1

It is well established that respiratory organisms use proton motive force to produce ATP via F-type ATP synthase aerobically and that this process may reverse during anaerobiosis to produce proton motive force. Here, we show that Shewanella oneidensis strain MR-1, a nonfermentative, facultative anaerobe known to respire exogenous electron acceptors, generates ATP primarily from substrate-level phosphorylation under anaerobic conditions. Mutant strains lacking ackA (SO2915) and pta (SO2916), genes required for acetate production and a significant portion of substrate-level ATP produced anaerobically, were tested for growth. These mutant strains were unable to grow anaerobically with lactate and fumarate as the electron acceptor, consistent with substrate-level phosphorylation yielding a significant amount of ATP. Mutant strains lacking ackA and pta were also shown to grow slowly using N-acetylglucosamine as the carbon source and fumarate as the electron acceptor, consistent with some ATP generation deriving from the Entner-Doudoroff pathway with this substrate. A deletion strain lacking the sole F-type ATP synthase (SO4746 to SO4754) demonstrated enhanced growth on N-acetylglucosamine and a minor defect with lactate under anaerobic conditions. ATP synthase mutants grown anaerobically on lactate while expressing proteorhodopsin, a light-dependent proton pump, exhibited restored growth when exposed to light, consistent with a proton-pumping role for ATP synthase under anaerobic conditions. Although S. oneidensis requires external electron acceptors to balance redox reactions and is not fermentative, we find that substrate-level phosphorylation is its primary anaerobic energy conservation strategy. Phenotypic characterization of an ackA deletion in Shewanella sp. strain MR-4 and genomic analysis of other sequenced strains suggest that this strategy is a common feature of Shewanella.Shewanella oneidensis strain MR-1 is a nonfermentative, facultative anaerobe which respires various substrates, including oxygen, soluble metals, insoluble iron and manganese oxide minerals, electrodes, and organic compounds (8, 12, 18, 22). Other bacteria with the ability to respire electrodes and oxide minerals, such as Geobacter and Geothrix, oxidize acetate to carbon dioxide (4, 7, 9), consistent with these organisms generating ATP primarily from oxidative phosphorylation rather than substrate-level phosphorylation. Yet, an examination of metabolic end products and a variety of central metabolism and flux analyses of MR-1 show that acetate is the major product under anaerobic conditions (18, 27, 29, 31). The general anaerobic metabolism model for MR-1, as depicted in Fig. ​Fig.1,1, has key features of glycolysis via the Entner-Doudoroff pathway as well as acetyl coenzyme A (acetyl-CoA) flux toward acetate anaerobically via phosphate acetyltransferase (Pta) and acetate kinase (AckA) (27, 29, 31). High-performance liquid chromatography (HPLC) studies in our lab and others have shown that pyruvate may be excreted during lactate utilization both aerobically and anaerobically (30, 31), and MR-1 has not been shown to maintain significant flux through the tricarboxylic acid (TCA) cycle under anaerobic conditions (31).Open in a separate windowFIG. 1.Simplified model of S. oneidensis central metabolism. Entner-Doudoroff glycolysis yields two molecules of pyruvate. Under aerobic conditions, pyruvate facilitates the reduction of NAD⁺ to NADH before being completely oxidized to carbon dioxide in the TCA cycle. Anaerobically, pyruvate oxidation to acetyl-CoA yields formate before the pyruvate is converted to acetate. Formate is subsequently oxidized to carbon dioxide. Reactions catalyzed by acetate kinase and phosphate acetyltransferase are denoted AckA and Pta, respectively. QH₂ is reduced quinone. The model is based on several references (5, 24, 29, 31, 36).Characterization studies of proton motive force (PMF) in MR-1 have not definitively determined whether the source of anaerobic proton pumping or translocation is electron transport, ATP synthase, or metabolite transport (13, 19). Myers et al. demonstrated that anaerobic MR-1 cells starved of electron acceptor generate PMF in response to fumarate addition (19). However, the directionality of the ATP synthase (i.e., generation of ATP or ATPase to pump protons) was not characterized. Previous work has confirmed that proteorhodopsin (PR), a light-dependent, proton-pumping integral membrane protein, can be used in MR-1 to supplement PMF (13). However, the observed increase in PMF in wild-type cells expressing PR did not result in higher optical densities (ODs) or in a higher growth rate. Though all known bacteria depend on PMF, whether MR-1 uses that PMF for ATP production or uses ATP to help generate PMF under anaerobic conditions has yet to be determined.To examine ATP production in MR-1, growth on carbon sources that offer various amounts of substrate-level-derived ATP and reducing equivalents (NADH, formate, or quinones) in their oxidation was characterized. Two carbon sources entering central metabolism at different locations are N-acetylglucosamine (NAG) and lactate, which enter before and after glycolysis, respectively (Fig. ​(Fig.1)1) (24, 36). Both are oxidized to acetate and carbon dioxide anaerobically, though lactate yields one ATP and two reducing equivalents per molecule, while NAG yields three ATPs and four reducing equivalents per molecule. The differences in ATP yields derived from utilization of NAG versus lactate, combined with modification of those yields through gene deletions, allowed for characterization of ATP production in MR-1.The goal of this work was to elucidate the primary source of ATP generation under anaerobic conditions in MR-1. Data presented here support a model of anaerobic metabolism where substrate-level phosphorylation is the primary mechanism for ATP generation and where some amount of the ATP pool is used to generate PMF. Paradoxically, the most diverse respiratory organism characterized to date (8, 12, 22) does not generate ATP from electron transport reactions and PMF. Our finding highlights a critical difference in metabolic strategies between Shewanella and other organisms that are able to reduce insoluble substrates, such as Geobacter and Geothrix.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability,Localization, and Subsequent Budding of Virus-Like Particles

The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the ₉₆LPLGVA₁₀₁ sequence of eVP40 and the corresponding ₈₄LPLGIM₈₉ sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the ₉₆LPLGVA₁₀₁ motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the ₉₆LPLGVA₁₀₁ motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.Ebola and Marburg viruses are members of the family Filoviridae. Filoviruses are filamentous, negative-sense, single-stranded RNA viruses that cause lethal hemorrhagic fevers in both humans and nonhuman primates (5). Filoviruses encode seven viral proteins including: NP (major nucleoprotein), VP35 (phosphoprotein), VP40 (matrix protein), GP (glycoprotein), VP30 (minor nucleoprotein), VP24 (secondary matrix protein), and L (RNA-dependent RNA polymerase) (2, 5, 10, 12, 45). Numerous studies have shown that expression of Ebola virus VP40 (eVP40) alone in mammalian cells leads to the production of virus-like particles (VLPs) with filamentous morphology which is indistinguishable from infectious Ebola virus particles (12, 17, 18, 25, 26, 27, 30, 31, 34, 49). Like many enveloped viruses such as rhabdovirus (11) and arenaviruses (44), Ebola virus encodes late-assembly or L domains, which are sequences required for the membrane fission event that separates viral and cellular membranes to release nascent virion particles (1, 5, 7, 10, 12, 18, 25, 27, 34). Thus far, four classes of L domains have been identified which were defined by their conserved amino acid core sequences: the Pro-Thr/Ser-Ala-Pro (PT/SAP) motif (25, 27), the Pro-Pro-x-Tyr (PPxY) motif (11, 12, 18, 19, 41, 53), the Tyr-x-x-Leu (YxxL) motif (3, 15, 27, 37), and the Phe-Pro-Ile-Val (FPIV) motif (39). Both PTAP and the PPxY motifs are essential for efficient particle release for eVP40 (25, 27, 48, 49), whereas mVP40 contains only a PPxY motif. L domains are believed to act as docking sites for the recruitment of cellular proteins involved in endocytic trafficking and multivesicular body biogenesis to facilitate virus-cell separation (8, 13, 14, 16, 28, 29, 33, 36, 43, 50, 51).In addition to L domains, oligomerization, and plasma-membrane localization of VP40 are two functions of the protein that are critical for efficient budding of VLPs and virions. Specific sequences involved in self-assembly and membrane localization have yet to be defined precisely. However, recent reports have attempted to identify regions of VP40 that are important for its overall function in assembly and budding. For example, the amino acid region ₂₁₂KLR₂₁₄ located at the C-terminal region was found to be important for efficient release of eVP40 VLPs, with Leu₂₁₃ being the most critical (30). Mutation of the ₂₁₂KLR₂₁₄ region resulted in altered patterns of cellular localization and oligomerization of eVP40 compared to those of the wild-type genotype (30). In addition, the proline at position 53 was also implicated as being essential for eVP40 VLP release and plasma-membrane localization (54).In a more recent study, a YPLGVG motif within the M protein of Nipah virus (NiV) was shown to be important for stability, membrane binding, and budding of NiV VLPs (35). Whether this NiV M motif represents a new class of L domain remains to be determined. However, it is clear that this YPLGVG motif of NiV M is important for budding, perhaps involving a novel mechanism (35). Our rationale for investigating the corresponding, conserved motifs present within the Ebola and Marburg virus VP40 proteins was based primarily on these findings with NiV. In addition, Ebola virus VP40 motif maps close to the hinge region separating the N- and C-terminal domains of VP40 (4). Thus, the ₉₆LPLGVA₁₀₁ motif of eVP40 is predicted to be important for the overall stability and function of VP40 during egress. Findings presented here indicate that disruption of these filovirus VP40 motifs results in a severe defect in VLP budding, due in part to impairment in overall VP40 structure, stability and/or intracellular localization.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Effect of Biowaste Sludge Maturation on the Diversity of Thermophilic Bacteria and Archaea in an Anaerobic Reactor

Prokaryotic diversity was investigated near the inlet and outlet of a plug-flow reactor. After analyzing 800 clones, 50 bacterial and 3 archaeal phylogenetic groups were defined. Clostridia (>92%) dominated among bacteria and Methanoculleus (>90%) among archaea. Significant changes in pH and volatile fatty acids did not invoke a major shift in the phylogenetic groups. We suggest that the environmental filter imposed by the saline conditions (20 g liter⁻¹) selected a stable community of halotolerant and halophilic prokaryotes.The anaerobic digestion of organic wastes constitutes a major research focus due to the global needs for waste recycling and renewable energy production. Currently, the linkage between digester performance and the diversity and dynamics of anaerobic prokaryotes is still not fully understood (2). Bacterial diversity in anaerobic reactors has always been judged to be greater than archaeal diversity (9, 13, 30). This probably reflects the metabolic flexibility of bacteria and the range of available substrates in complex input materials. However, several recent discoveries pose the question as to whether archaeal diversity and physiological versatility are greater than currently thought: that is, the huge diversity of yet-to-be cultured archaea (4, 6), the detection of energy metabolisms not known previously in archaea (e.g., chemoorganotrophy [1]), and the unexpected predominance of archaeal groups among prokaryotes in unstressed environments, such as ammonia oxidizers in soils (19).Several surveys have investigated the shifts in prokaryotic diversity occurring with waste maturation or under different reactor operating conditions. Some evidence demonstrates bacterial phylogenetic stability under constant operation conditions (18). Generally, however, the dominant bacterial communities are very dynamic, showing chaotic shifts even with stable reactor performance (9, 32). Hypothetically, this is due to the functional redundancy among diverse phylogenetic groups allowing oscillations of their populations with no effects on the reactor function (2). Archaeal communities are less dynamic than bacterial communities (32), their shifts being related to changes in reactor performance (6) and correlated with important process parameters such as volatile fatty acids (VFAs) (13, 16).We aimed to analyze the change in prokaryotic diversity in a plug-flow reactor associated with the maturation of biowastes. In a previous study, stable bacterial and archaeal denaturing gradient gel electrophoresis patterns were found in the sludge collected close to the outlet over a year of unstable reactor performance (23). This temporal pattern contradicts the general idea of extremely dynamic bacterial communities proliferating in bioreactors. Here, we investigated the phylogenetic identity of the organisms in sludge samples collected near the inlet and outlet pipes after a period of stable operation and performance in terms of pH and biogas production.     

Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization

Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.The ability of a cell to rapidly respond to extracellular cues and direct cytoskeletal rearrangements is dependent on an array of signaling complexes that control actin assembly (58). The protrusive structures at the leading edges of motile cells are broadly defined as lamellipodia or filopodia (14). Lamellae are sheet-like protrusions composed of dendritic actin arrays that drive membrane expansion, with the “lamellipodium” representing a narrow region at the edge of the cell (in culture) characterized by rapid actin polymerization. This F-actin assembly is suggested to require Arp2/3 activity that nucleates new actin filaments from the sides of existing ones (58, 71) and capping proteins that limit the length of these new filaments and stabilize them (7). Arp2/3 activity in turn is regulated by the WASP/WAVE family of proteins, such as N-WASP and WAVE2 (68), whose regulation is a subject of intense interest (12, 29, 36, 41, 56, 76).Filopodia contain parallel bundles of actin filaments containing fascin (22). These are dynamic structures that emanate from the periphery of the cell and are retracted, with occasional attachment (to the dish in culture). Thus, they have been thought to have a sensory or exploratory role during cell migration (28). This is the case for neuronal growth cones, where filopodia sense attractant or repulsive cues and dictate direction in axonal path finding (9, 17, 25, 35). Filopodia have been shown to be important in the context of dendritic-spine development (64, 77), epithelial-sheet closure (26, 60, 79), and cell invasion/metastasis (80, 83).Lamellipodia have been well characterized since the pioneering work of Abercrombie et al. in the early 1970s (2, 3, 4). Filopodia require symmetry breaking at the leading edge (initiation), followed by elongation driven by a filopodial-tip protein complex (14, 28). A few proteins have been identified in this complex; Mena/Vasp serve to prevent capping at the barbed ends of bundled actin filaments (7, 53), and Dia2 promotes F-actin elongation (57, 85). Termination of filopodial elongation is not understood but nonetheless is likely to be tightly regulated. In the absence of F-actin elongation, retraction of the filopodium takes place by a rearward flow of F-actin and filament depolymerization (22).IRSp53 is in a position to play a pivotal role in generating filopodia; this brain-enriched protein was discovered as a substrate of the insulin receptor (87). Subsequently, IRSp53 was identified as an effector for Rac1 (50) and Cdc42 (27, 38), where it participates in filopodium and lamellipodium production (38, 51, 54, 86), neurite extension (27), dendritic-spine morphogenesis (1, 15, 66, 67), cell motility and invasiveness (24). The N terminus of IRSp53 contains a conserved helical domain that is found in five different gene products and is referred to as the IRSp53/MIM homology domain (IMD) (51, 70). This domain has been postulated to bind to Rac1 (50, 70) in a nucleotide-independent manner (52), but no convincing effector-like region has been identified. A Cdc42-specific CRIB-like sequence that does not bind Rac1 (27, 38) allows coupling of this and perhaps related Rho GTPases. The structure of the IMD reveals a zeppelin-shaped dimer that could bind “bent” membranes; thus, its potential as an F-actin-bundling domain (51, 82) could be an in vitro artifact often attributed to proteins with basic patches (46). Although there are reports of F-actin binding at physiological ionic strength (ca. 100 mM KCl) (82, 19), this region when expressed in isolation does not decorate F-actin in vivo.Two reports showed the IMD to be an “inverse-BAR” domain. BAR (Bin-amphipysin-Rvs) domains are found in proteins involved in endocytic trafficking, such as amphipysin and endophilin, and stabilize positively bent membranes, such as those on endocytic vesicles (31, 47). The IMD domains of both IRSp53 (70) and MIM-B (46) associate with lipids and can induce tubulations of PI(3,4,5)P₃ or PI(4,5)P₂-rich membranes, respectively. These tubulations are equivalent to membrane protrusions and are also referred to as negatively bent membranes. Ectopic expression of the IMD from IRSp53 (51, 70, 82, 86) or two other family members, MIM-B (11, 46) and IRTKS (52), can give rise to cells with many peripheral extensions. MIM-B is said to stimulate lamellipodia (11), while IRTKS generates “short actin clusters” at the cell periphery (52).In IRSp53 is a CRIB-like motif that mediates binding to Cdc42 (27, 38), but the function of this interaction in unclear. Cdc42 could relieve IRSp53 autoinhibition as described for N-Wasp (38), but there is little evidence for this. It has been suggested that Cdc42 controls IRSp53 localization and actin remodeling (27, 38), but another study indicated that these events are Cdc42 independent (19). IRSp53 contains a central SH3 domain that may bind proline-rich proteins, such as Dia1 (23), Mena (38), WAVE2 (49, 50, 69), and Eps8 (19, 24). However, it seems unlikely that all of these represent bona fide partners, and side-by-side comparison is provided in this study. Mena is involved in filopodium production (37), Dia1 in stress fiber formation (81), and WAVE2 in lamellipodium extension (72). Thus, Mena is a better candidate as a partner for IRSp53-mediated filopodia than Dia1 or WAVE2.There is good evidence for IRSp53 as a cellular partner for Eps8 (19). Eps8 is an adaptor protein containing an N-terminal PTB domain that can associate with receptor tyrosine kinases (65), and perhaps β integrins (13), and a C-terminal SH3 domain that can associate with Abi1 (30). Binding of the general adaptor Abi1 appears to positively regulate the actin-capping domain at the C terminus of Eps8 (18). It has been suggested that IRSp53 and Eps8 as a complex regulate cell motility, and perhaps Rac1 activation, via SOS (24); more recently, their roles in filopodium formation have been addressed (19). The involvement of IRSp53, but not MIM-B or IRTKS, in filopodium formation might be related to its role as a Cdc42 effector. We show here that, surprisingly, the CRIB motif is not essential for this activity, but rather, the ability of IRSp53 to associate via its SH3 domain is required, and that this domain is controlled by 14-3-3 binding.We have focused on the regulation of Cdc42 effectors that bind 14-3-3, including IRSp53 and PAK4, which are found as 14-3-3 targets in various proteomic projects (32, 44). In this study, we characterize the binding of 14-3-3 to IRSp53 and uncover how this activity regulates IRSp53 function. The phosphorylation-dependent 14-3-3 binding is GSK3β dependent, and 14-3-3 blocks the accessibility of both the CRIB and SH3 domains of IRSp53, thus indicating its primary function in controlling IRSp53 partners. This regulation of the SH3 domain by 14-3-3 is critical in the proper localization and termination of IRSp53 function to promote filopodium dynamics.