Effects of Aeration on the Synthesis of Poly(3-Hydroxybutyrate) from Glycerol and Glucose in Recombinant Escherichia coli

Bioreactor cultures of Escherichia coli recombinants carrying phaBAC and phaP of Azotobacter sp. FA8 grown on glycerol under low-agitation conditions accumulated more poly(3-hydroxybutyrate) (PHB) and ethanol than at high agitation, while in glucose cultures, low agitation led to a decrease in PHB formation. Cells produced smaller amounts of acids from glycerol than from glucose. Glycerol batch cultures stirred at 125 rpm accumulated, in 24 h, 30.1% (wt/wt) PHB with a relative molecular mass of 1.9 MDa, close to that of PHB obtained using glucose.Polyhydroxyalkanoates (PHAs), accumulated as intracellular granules by many bacteria under unfavorable conditions (5, 8), are carbon and energy reserves and also act as electron sinks, enhancing the fitness of bacteria and contributing to redox balance (9, 11, 19). PHAs have thermoplastic properties, are totally biodegradable by microorganisms present in most environments, and can be produced from different renewable carbon sources (8).Poly(3-hydroxybutyrate) (PHB) is the best known PHA, and its accumulation in recombinant Escherichia coli from several carbon sources has been studied (1, 13). In the last few years, increasing production of biodiesel has caused a sharp fall in the cost of its main by-product, glycerol (22). Its use for microbial PHA synthesis has been analyzed for natural PHA producers, such as Methylobacterium rhodesianum, Cupriavidus necator (formerly called Ralstonia eutropha) (3), several Pseudomonas strains (22), the recently described bacterium Zobellella denitrificans (7), and a Bacillus sp. (18), among others. Glycerol has also been used for PHB synthesis in recombinant E. coli (12, 15). PHAs obtained from glycerol were reported to have a significantly lower molecular weight than polymer synthesized from other substrates, such as glucose or lactose (10, 23).Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes that are involved in granule formation and/or have regulatory functions, such as phasins, granule-associated proteins that have been shown to enhance polymer synthesis and the number and size of PHA granules (17, 24). The phasin PhaP has been shown to exert a beneficial effect on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of strain K24KP, a recombinant E. coli that carries phaBAC and phaP of Azotobacter sp. FA8 (6).Because the redox state of the cells is known to affect the synthesis of PHB (1, 4, 14), the present study investigates the behavior of this recombinant strain under different aeration conditions, by using two substrates, glucose and glycerol, with different oxidation states.     

Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of Cronobacter Genomospecies (Enterobacter sakazakii) in Powdered Infant Formula

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.Cronobacter strains were originally described as Enterobacter sakazakii (12), but they are now known to comprise a novel genus consisting of six separate genomospecies (20, 21). These opportunistic pathogens are ubiquitous in the environment and various types of food and are occasionally found in the normal human flora (11, 12, 16, 32, 47). Based on case reports, Cronobacter infections in adults are generally less severe than Cronobacter infections in newborn infants, with which a high fatality rate is associated (24).The ability to detect Cronobacter and trace possible sources of infection is essential as a means of limiting the impact of these organisms on neonatal health and maintaining consumer confidence in powdered infant formula (PIF). Conventional methods, involving isolation of individual colonies followed by biochemical identification, are more time-consuming than molecular methods, and the reliability of some currently proposed culture-based methods has been questioned (28). Recently, several PCR-based techniques have been described (23, 26, 28-31, 38). These techniques are reported to be efficient even when low levels of Cronobacter cells are found in a sample (0.36 to 66 CFU/100 g). However, PCR requires DNA extraction and does not allow direct, in situ visualization of the bacterium in a sample.Fluorescence in situ hybridization (FISH) is a method that is commonly used for bacterial identification and localization in samples. This method is based on specific binding of nucleic acid probes to particular DNA or RNA target regions (1, 2). rRNA has been regarded as the most suitable target for bacterial FISH, allowing differentiation of potentially viable cells. Traditionally, FISH methods are based on the use of conventional DNA oligonucleotide probes, and a commercial system, VIT-E sakazakii (Vermicon A.G., Munich, Germany), has been developed based on this technology (25). However, a recently developed synthetic DNA analogue, peptide nucleic acid (PNA), has been shown to provide improved hybridization performance compared to DNA probes, making FISH procedures easier and more efficient (41). Taking advantage of the PNA properties, FISH using PNA has been successfully used for detection of several clinically relevant microorganisms (5, 15, 17, 27, 34-36).     

Fermentation of Glycerol to Succinate by Metabolically Engineered Strains of Escherichia coli

The fermentative metabolism of Escherichia coli was reengineered to efficiently convert glycerol to succinate under anaerobic conditions without the use of foreign genes. Formate and ethanol were the dominant fermentation products from glycerol in wild-type Escherichia coli ATCC 8739, followed by succinate and acetate. Inactivation of pyruvate formate-lyase (pflB) in the wild-type strain eliminated the production of formate and ethanol and reduced the production of acetate. However, this deletion slowed growth and decreased cell yields due to either insufficient energy production or insufficient levels of electron acceptors. Reversing the direction of the gluconeogenic phosphoenolpyruvate carboxykinase reaction offered an approach to solve both problems, conserving energy as an additional ATP and increasing the pool of electron acceptors (fumarate and malate). Recruiting this enzyme through a promoter mutation (pck*) to increase expression also increased the rate of growth, cell yield, and succinate production. Presumably, the high NADH/NAD⁺ ratio served to establish the direction of carbon flow. Additional mutations were also beneficial. Glycerol dehydrogenase and the phosphotransferase-dependent dihydroxyacetone kinase are regarded as the primary route for glycerol metabolism under anaerobic conditions. However, this is not true for succinate production by engineered strains. Deletion of the ptsI gene or any other gene essential for the phosphotranferase system was found to increase succinate yield. Deletion of pflB in this background provided a further increase in the succinate yield. Together, these three core mutations (pck*, ptsI, and pflB) effectively redirected carbon flow from glycerol to succinate at 80% of the maximum theoretical yield during anaerobic fermentation in mineral salts medium.Renewable bioenergy offers the potential to solve many environmental problems associated with petroleum-based fuels and chemicals. Biodiesel is produced by reacting vegetable oil or animal fat with alcohol (methanol or ethanol) and used as a transportation fuel in many countries (33). Glycerol is formed as an abundant waste product with limited commercial uses. As the worldwide production of biodiesel continues to increase, the development of effective uses for glycerol may prove essential for the economics and competitiveness of the biodiesel industry. The value of glycerol waste from biodiesel is similar to that of sugars currently used to produce fuel ethanol. Bioconversion of glycerol to higher-value products that replace petroleum, such as polymers, surfactants, solvents, and chemical intermediates, represents an opportunity to decrease waste and improve the economics of the biodiesel industry (5).Many previous investigations have focused on the fermentative production of 1,3-propanediol (1,3-PD) from glycerol (2, 26, 35). Microorganisms including Klebsiella (14), Citrobacter (6), Enterobacter (1), Lactobacillus (29), and Clostridium (10, 28) have the native ability to ferment glycerol into this product. Dupont and Genencor have commercialized a 1,3-PD-based polyester, a condensation product of 1,3-PD and terephthalic acid using glucose as the feedstock. Potential demand for this polymer is estimated to be 1 billion to 2 billion pounds per year over the next 10 years (26). Other investigations of glycerol fermentation have described the production of hydrogen and ethanol (15), polyhydroxyalkanoates (PHAs) (20, 27), glyceric acid (13), and small amounts of succinate (21).Succinic acid is currently used as a specialty chemical in the agricultural, food, and pharmaceutical industries (24, 34). It has also been identified by the U.S. Department of Energy as one of the top 12 building block chemicals (31) because it can be converted into a wide variety of products, including green solvents, pharmaceutical products, and biodegradable plastics (24, 34). Succinate is primarily produced from petroleum-derived maleic anhydride. Recent increases in the petroleum price have generated considerable interest in the fermentative production of succinate from sugars using either natural succinate-producing rumen bacteria or metabolically engineered Escherichia coli strains (24, 36, 38). Succinate can also be produced from glycerol by rumen bacteria, such as Anaerobiospirillum succiniciproducens (21). However, these strains require complex nutrients that increase costs of production, purification, and waste treatment.E. coli has been previously engineered for the commercial production of 1,3-PD from sugars by Dupont and Genecor (26). It is an excellent organism for biotechnology applications but was long thought incapable of anaerobic growth on glycerol (23). Recent studies demonstrated that E. coli can ferment glycerol anaerobically (8, 11, 25, 33), and a new model was proposed for glycerol fermentation (11). In this model, glycerol is metabolized through the glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (encoded by dhaKLM) pathway with the production of ethanol and acetate as primary fermentation products (11). Small amounts of succinate and 1,2-propanediol were also produced. Native genes encoding glycerol dehydrogenase and dihydroxyacetone kinase were expressed from a plasmid to increase the rates of glycerol metabolism and ethanol production (32). Succinate production has also been increased by expressing Clostridium freundii dihydroxyacetone kinase (encoded by dhaKL) (11). However, neither of these enhanced pathways would appear suitable for efficient succinate production due to the absence of net ATP production and the requirement for phosphoenolpyruvate as a phosphoryl donor for dihydroxyacetone, limiting the carboxylation of this intermediate (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Glycerol uptake and fermentation by E. coli. (A) Native E. coli pathways. Bold black arrows represent dominant fermentation reactions prior to engineering; thin black arrows represent minor fermentation reactions. GlpK and GlpD are thought to function primarily during aerobic metabolism. Pathways are based on current reviews in EcoSal (3, 4, 22), data available in Ecocyc (19), and primary literature (11, 12, 18, 25, 30). (B) Engineered pathway for the fermentative metabolism of glycerol to succinate. Bold black arrows represent the engineered reactions for glycerol fermentation to succinate as the dominant product; thin black arrows represent minor fermentation reactions in the engineered strain. Dashed arrows represent reactions that are not functional due to deletions in ptsI and pflB. Deleted genes are marked with a black X. In native E. coli strains, phosphoenolpyruvate carboxykinase functions during gluconeogenesis to produce phosphoenolpyruvate. Mutational activation of the pck gene (denoted pck*) allows this enzyme to function in the reverse direction and to serve as the dominant carboxylation step, conserving energy as ATP. With this engineered pathway, competing needs for PEP have been eliminated and net ATP production has been increased. PEP is boxed to indicate a common pool. Abbreviations: DHA, dihydroxyacetone; DHAP, dihydroxyacetone 3-phosphate; PEP, phosphoenolpyruvate; G3P, glycerol 3-phosphate; GA3P, glyceraldehydes 3-phosphate.Previous studies in our laboratory (16, 17, 36, 38) have engineered E. coli ATCC 8739 for the efficient production of succinate from glucose by recruiting genes from alternative pathways (36, 38). In this paper, we report the use of a similar approach to engineer strains for succinate production from glycerol in mineral salts medium.     

Role of Geobacter sulfurreducens Outer Surface c-Type Cytochromes in Reduction of Soil Humic Acid and Anthraquinone-2,6-Disulfonate

Deleting individual genes for outer surface c-type cytochromes in Geobacter sulfurreducens partially inhibited the reduction of humic substances and anthraquinone-2,6,-disulfonate. Complete inhibition was obtained only when five of these genes were simultaneously deleted, suggesting that diverse outer surface cytochromes can contribute to the reduction of humic substances and other extracellular quinones.Humic substances can play an important role in the reduction of Fe(III), and possibly other metals, in sedimentary environments (6, 34). Diverse dissimilatory Fe(III)-reducing microorganisms (3, 5, 7, 9, 11, 19-22, 25) can transfer electrons onto the quinone moieties of humic substances (38) or the model compound anthraquinone-2,6-disulfonate (AQDS). Reduced humic substances or AQDS abiotically reduces Fe(III) to Fe(II), regenerating the quinone. Electron shuttling in this manner can greatly increase the rate of electron transfer to insoluble Fe(III) oxides, presumably because soluble quinone-containing molecules are more accessible for microbial reduction than insoluble Fe(III) oxides (19, 22). Thus, catalytic amounts of humic substances have the potential to dramatically influence rates of Fe(III) reduction in soils and sediments and can promote more rapid degradation of organic contaminants coupled to Fe(III) reduction (1, 2, 4, 10, 24).To our knowledge, the mechanisms by which Fe(III)-reducing microorganisms transfer electrons to humic substances have not been investigated previously for any microorganism. However, reduction of AQDS has been studied using Shewanella oneidensis (17, 40). Disruption of the gene for MtrB, an outer membrane protein required for proper localization of outer membrane cytochromes (31), inhibited reduction of AQDS, as did disruption of the gene for the outer membrane c-type cytochrome, MtrC (17). However, in each case inhibition was incomplete, and it was suggested that there was a possibility of some periplasmic reduction (17), which would be consistent with the ability of AQDS to enter the cell (40).The mechanisms for electron transfer to humic substances in Geobacter species are of interest because molecular studies have frequently demonstrated that Geobacter species are the predominant Fe(III)-reducing microorganisms in sedimentary environments in which Fe(III) reduction is an important process (references 20, 32, and 42 and references therein). Geobacter sulfurreducens has routinely been used for investigations of the physiology of Geobacter species because of the availability of its genome sequence (29), a genetic system (8), and a genome-scale metabolic model (26) has made it possible to take a systems biology approach to understanding the growth of this organism in sedimentary environments (23).     

Abundance of Novel and Diverse tfdA-Like Genes,Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases,in Soil

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by α-ketoglutarate- and Fe²⁺-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdAα). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of tfdA-like genes in soil. Five primer sets targeting tfdA-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified tfdA-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated tfdA-like genes indicated that soil tfdA-like genes were related to those of group 2 and 3 Bradyrhizobium spp., Sphingomonas spp., and uncultured soil bacteria. Soil-derived tfdA-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse tfdA-like genes. Correlation analysis of 16S rRNA and tfdA-like gene similarity indicated that any two bacteria with D > 20% of group 2 tfdA-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2 tfdA-like genes. Novel qPCR assays were established to quantify such new tfdA-like genes. Copy numbers of tfdA-like genes were 1.0 × 10⁶ to 65 × 10⁶ per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.Phenoxyalkanoic acid (PAA) herbicides such as MCPA (4-chloro-2-methyl-phenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are widely used to control broad-leaf weeds in agricultural as well as nonagricultural areas (19, 77). Degradation occurs primarily under oxic conditions in soil, and microorganisms play a key role in the degradation of such herbicides in soil (62, 64). Although relatively rapidly degraded in soil (32, 45), both MCPA and 2,4-D are potential groundwater contaminants (10, 56, 70), accentuating the importance of bacterial PAA herbicide-degrading bacteria in soils (e.g., references 3, 5, 6, 20, 41, 59, and 78).Degradation can occur cometabolically or be associated with energy conservation (15, 54). The first step in the degradation of 2,4-D and MCPA is initiated by the product of cadAB or tfdA-like genes (29, 30, 35, 67), which constitutes an α-ketoglutarate (α-KG)- and Fe²⁺-dependent dioxygenase. TfdA removes the acetate side chain of 2,4-D and MCPA to produce 2,4-dichlorophenol and 4-chloro-2-methylphenol, respectively, and glyoxylate while oxidizing α-ketoglutarate to CO₂ and succinate (16, 17).Organisms capable of PAA herbicide degradation are phylogenetically diverse and belong to the Alpha-, Beta-, and Gammproteobacteria and the Bacteroidetes/Chlorobi group (e.g., references 2, 14, 29-34, 39, 60, 68, and 71). These bacteria harbor tfdA-like genes (i.e., tfdA or tfdAα) and are categorized into three groups on an evolutionary and physiological basis (34). The first group consists of beta- and gammaproteobacteria and can be further divided into three distinct classes based on their tfdA genes (30, 46). Class I tfdA genes are closely related to those of Cupriavidus necator JMP134 (formerly Ralstonia eutropha). Class II tfdA genes consist of those of Burkholderia sp. strain RASC and a few strains that are 76% identical to class I tfdA genes. Class III tfdA genes are 77% identical to class I and 80% identical to class II tfdA genes and linked to MCPA degradation in soil (3). The second group consists of alphaproteobacteria, which are closely related to Bradyrhizobium spp. with tfdAα genes having 60% identity to tfdA of group 1 (18, 29, 34). The third group also harbors the tfdAα genes and consists of Sphingomonas spp. within the alphaproteobacteria (30).Diverse PAA herbicide degraders of all three groups were identified in soil by cultivation-dependent studies (32, 34, 41, 78). Besides CadAB, TfdA and certain TfdAα proteins catalyze the conversion of PAA herbicides (29, 30, 35). All groups of tfdA-like genes are potentially linked to the degradation of PAA herbicides, although alternative primary functions of group 2 and 3 TfdAs have been proposed (30, 35). However, recent cultivation-independent studies focused on 16S rRNA genes or solely on group 1 tfdA sequences in soil (e.g., references 3-5, 13, and 41). Whether group 2 and 3 tfdA-like genes are also quantitatively linked to the degradation of PAA herbicides in soils is unknown. Thus, tools to target a broad range of tfdA-like genes are needed to resolve such an issue. Primers used to assess the diversity of tfdA-like sequences used in previous studies were based on the alignment of approximately 50% or less of available sequences to date (3, 20, 29, 32, 39, 47, 58, 73). Primers specifically targeting all major groups of tfdA-like genes to assess and quantify a broad diversity of potential PAA degraders in soil are unavailable. Thus, the objectives of this study were (i) to develop primers specific for all three groups of tfdA-like genes, (ii) to establish quantitative kinetic PCR (qPCR) assays based on such primers for different soil samples, and (iii) to assess the diversity and abundance of tfdA-like genes in soil.     

Escherichia coli Strains Engineered for Homofermentative Production of d-Lactic Acid from Glycerol

Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to d-lactic acid (d-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for d-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of d-lactate from pyruvate (d-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (ΔpflB) and fumarate reductase (ΔfrdA) (strain LA01) and (ii) inactivation of fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and alcohol/acetaldehyde dehydrogenase (ΔadhE) (strain LA02). A mutation that blocked the aerobic d-lactate dehydrogenase (Δdld) also was introduced in both LA01 and LA02 to prevent the utilization of d-lactate. The most efficient strain (LA02Δdld, with GlpK-GlpD overexpressed) produced 32 g/liter of d-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for d-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of d-lactate and is redox balanced, thus representing a viable metabolic pathway.Lactic acid (lactate) and its derivatives have many applications in the food, pharmaceutical, and polymer industries (13, 30). An example is polylactic acid, a renewable, biodegradable, and environmentally friendly polymer produced from d- and l-lactate (19). In this context, biological processes have the advantage of being able to produce chirally pure lactate from inexpensive media containing only the carbon source and mineral salts (43). While lactic acid bacteria traditionally have been used in the production of d-lactate from carbohydrate-rich feedstocks, several laboratories recently have reported alternative biocatalysts (13, 30), many of which are engineered Escherichia coli strains that produce d- or l-lactate (4, 8, 50, 51, 52).Unlike the aforementioned reports, which have dealt with the use of carbohydrates, our work focuses on the use of glycerol as a carbon source for the production of d-lactate. Glycerol has become an inexpensive and abundant substrate due to its generation in large amounts as a by-product of biodiesel and bioethanol production (18, 32, 47). The conversion of glycerol to higher-value products has been proposed as a path to economic viability for the biofuels industry (47). One such product is lactate, whose production could be readily integrated into existing biodiesel and bioethanol facilities, thus establishing true biorefineries.Although many microorganisms are able to metabolize glycerol (25), the use of industrial microbes such as E. coli could greatly accelerate the development of platforms to produce fuels and chemicals from this carbon source. We recently reported on the ability of E. coli to metabolize glycerol under either anaerobic or microaerobic conditions and identified the environmental and metabolic determinants of these processes (9, 11, 28). In one of the studies, the pathways involved in the microaerobic utilization of glycerol were elucidated, and they are shown in Fig. ​Fig.11 (9). A common characteristic of glycerol metabolism under either anaerobic or microaerobic conditions is the generation of ethanol as the primary product and the negligible production of lactate (6, 9, 11, 28). In the work reported here, the knowledge base created by the aforementioned studies was used to engineer E. coli for the efficient conversion of glycerol to d-lactate in minimal medium. The engineered strains hold great promise as potential biocatalysts for the conversion of low-value glycerol streams to a higher-value product like d-lactate.Open in a separate windowFIG. 1.Pathways involved in the microaerobic utilization of glycerol in E. coli (9). Genetic modifications supporting the metabolic engineering strategies employed in this work are illustrated by thicker lines (overexpression of gldA-dhaKLM, glpK-glpD, and ldhA) or cross bars (disruption of pflB, pta, adhE, frdA, and dld). Broken lines illustrate multiple steps. Relevant reactions are represented by the names of the gene(s) coding for the enzymes: aceEF-lpdA, pyruvate dehydrogenase complex; adhE, acetaldehyde/alcohol dehydrogenase; ackA, acetate kinase; dhaKLM, dihydroxyacetone kinase; dld, respiratory d-lactate dehydrogenase; fdhF, formate dehydrogenase, part of the formate hydrogenlyase complex; frdABCD, fumarate reductase; gldA, glycerol dehydrogenase; glpD, aerobic glycerol-3-phosphate dehydrogenase; glpK, glycerol kinase; hycB-I, hydrogenase 3, part of the formate hydrogenlyase complex; ldhA, fermentative d-lactate dehydrogenase; pflB, pyruvate formate-lyase; pta, phosphate acetyltransferase; pykF, pyruvate kinase. Abbreviations: DHA, dihydroxyacetone; DHAP, DHA phosphate; G-3-P, glycerol-3-phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; P/O, amount of ATP produced in the oxidative phosphorylation per pair of electrons transferred through the electron transport system; QH₂, reduced quinones.     

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).     

Inhibiting the Initiation of Clostridium difficile Spore Germination using Analogs of Chenodeoxycholic Acid,a Bile Acid

To cause disease, Clostridium difficile spores must germinate in the host gastrointestinal tract. Germination is initiated upon exposure to glycine and certain bile acids, e.g., taurocholate. Chenodeoxycholate, another bile acid, inhibits taurocholate-mediated germination. By applying Michaelis-Menten kinetic analysis to C. difficile spore germination, we found that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination and appears to interact with the spores with greater apparent affinity than does taurocholate. We also report that several analogs of chenodeoxycholate are even more effective inhibitors. Some of these compounds resist 7α-dehydroxylation by Clostridium scindens, a core member of the normal human colonic microbiota, suggesting that they are more stable than chenodeoxycholate in the colonic environment.Clostridium difficile is a Gram-positive, spore-forming, anaerobic bacterium that is pathogenic for both humans and animals (33, 44). Infections caused by C. difficile range from mild diarrhea to more life-threatening conditions, such as pseudomembranous colitis (33). In the classic case, prior antibiotic treatment that disrupts the normally protective colonic flora makes patients susceptible to C. difficile infection (CDI) (35, 53). Other antibiotics, such as vancomycin and metronidazole, are the most commonly used treatments for CDI (54). However, because these antibiotics also disrupt the colonic flora, 10 to 40% of patients whose symptoms have been ameliorated suffer from relapsing CDI (15, 24). The annual treatment-associated cost for CDI in the United States is estimated to be between $750 million and $3.2 billion (8, 9, 16, 31). Moreover, the number of fatal cases of CDI has been increasing rapidly (14, 39). Thus, there is an urgent need to find alternative therapies for CDI.C. difficile infection likely is initiated by infection with the spore form of C. difficile (12). C. difficile elicits disease through the actions of two secreted toxins, TcdA and TcdB (48). TcdB was recently shown to be critical for pathogenesis in an animal model of disease (18). Since the toxins are produced by vegetative cells, not by spores (17), germination and outgrowth are prerequisites for pathogenesis.Spore germination is triggered by the interaction of small molecules, called germinants, with receptors within the spore inner membrane. These germinants vary by bacterial species and can include ions, amino acids, sugars, nucleotides, surfactants, or combinations thereof (43). The recognition of germinants triggers irreversible germination, leading to Ca²⁺-dipicolinic acid release, the uptake of water, the degradation of the cortex, and, eventually, the outgrowth of the vegetative bacterium (43). The germination receptors that C. difficile uses to sense the environment have not been identified. Based on homology searches, C. difficile germination receptors must be very different from known germination receptors (42), but they appear to be proteinaceous (13).Taurocholate, a primary bile acid, has been used for approximately 30 years by researchers and clinical microbiologists to increase colony formation by C. difficile spores from patient and environmental samples (3, 49, 51, 52). This suggested that C. difficile spores interact with bile acids along the gastrointestinal (GI) tract and that spores use a host-derived signal to initiate germination.The liver synthesizes the two major primary bile acids, cholate and chenodeoxycholate (40). These compounds are modified by conjugation with either taurine (to give taurocholate or taurochenodeoxycholate) or glycine (producing glycocholate or glycochenodeoxycholate). Upon secretion into the digestive tract, bile aids in the absorption of fat and cholesterol; much of the secreted bile is actively absorbed and recycled back to the liver for reutilization (40). Though efficient, enterohepatic recirculation is not complete; bile enters the cecum of the large intestine at a concentration of approximately 2 mM (30).In the cecum, bile is modified by the normal, benign colonic flora. First, bile salt hydrolases found on the surfaces of many bacterial species remove the conjugated amino acid, producing the deconjugated primary bile acids cholate and chenodeoxycholate (40). These deconjugated primary bile acids are further metabolized by only a few species of intestinal bacteria, including Clostridium scindens. C. scindens actively transports unconjugated primary bile acids into the cytoplasm, where they are 7α-dehydroxylated, converting cholate to deoxycholate and chenodeoxycholate to lithocholate (21, 40). The disruption of the colonic flora by antibiotic treatment abolishes 7α-dehydroxylation activity (41).Building upon the work on Wilson and others (51, 52), we demonstrated that taurocholate and glycine, acting together, trigger the loss of the birefringence of C. difficile spores (45). All cholate derivatives (taurocholate, glycocholate, cholate, and deoxycholate) stimulate the germination of C. difficile spores (45). Recently it was shown that taurocholate binding is prerequisite to glycine binding (37). At physiologically relevant concentrations, chenodeoxycholate inhibits taurocholate-mediated germination (46) and also inhibits C. difficile vegetative growth, as does deoxycholate (45). In fact, C. difficile spores use the relative concentrations of the various bile acids as cues for germination within the host (10).Since chenodeoxycholate is absorbed by the colonic epithelium and metabolized to lithocholate by the colonic flora (25, 40), the use of chenodeoxycholate as a therapy against C. difficile disease might be hindered by its absorption and conversion to lithocholate.Here, we further characterize the interaction of C. difficile spores with various bile acids and demonstrate that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination. Further, we identify chemical analogs of chenodeoxycholate that are more potent inhibitors of germination and that resist 7α-dehydroxylation by the colonic flora, potentially increasing their stability and effectiveness as inhibitors of C. difficile spore germination in the colonic environment.     

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.     

YjhS (NanS) Is Required for Escherichia coli To Grow on 9-O-Acetylated N-Acetylneuraminic Acid

The nanATEK-yhcH, yjhATS, and yjhBC operons in Escherichia coli are coregulated by environmental N-acetylneuraminic acid, the most prevalent sialic acid in nature. Here we show that YjhS (NanS) is a probable 9-O-acetyl N-acetylneuraminic acid esterase required for E. coli to grow on this alternative sialic acid, which is commonly found in mammalian host mucosal sites.The coregulated nanATEK-yhcH, yjhATS, and yjhBC operons involved in sialic acid catabolism in Escherichia coli are thought to be induced by the most common sialic acid, N-acetylneuraminic acid (Neu5Ac), through reversible inactivation of the NanR repressor encoded by nanR mapping immediately upstream of nanA (15, 27, 28; http://vetmed.illinois.edu/path/sialobiology/). Sialic acids are a family of over 40 naturally occurring 9-carbon keto sugar acids found mainly in metazoans of the deuterostome (starfish to human) developmental lineage and in some, mostly pathogenic, bacteria, where sialic acids expressed at the microbial cell surface inhibit host innate immunity (27). By contrast, most bacterial commensals and pathogens catabolize sialic acids as sole carbon and nitrogen sources, indicating exploitation of the sialic acid-rich host mucosal environment by a wide range of species (2, 27, 28). Interestingly, in vivo experimental evidence further indicates that sialic acid catabolism functions directly (nutrition) or indirectly (surface decoration and cell signaling) in host-microbe commensal and pathogenic interactions in organisms such as E. coli, Haemophilus influenzae, Pasteurella multocida, Salmonella enterica serovar Typhi, Streptococcus pneumoniae, Vibrio vulnificus, and Vibrio cholerae (1, 3, 5, 6, 10, 14, 23, 24, 26, 29). The animal species used for these studies include rodent models and natural hosts such as cattle and turkeys. The structural diversity of sialic acids at the terminal positions on glycoconjugates (glycoproteins and glycolipids) of mucosal surfaces of these hosts requires sialidases, acetyl esterases, and probably other enzymes that convert alternative or at least minor sialic acids to the more digestible Neu5Ac form (8, 9). We have previously demonstrated that E. coli has an epicurean propensity for metabolizing alternative sialic acids (30, 31). In the current communication, we show that YjhS is required for growth of E. coli on 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac₂).Because most sialic acids are bound to other sugars, including other sialic acids, as part of the oligosaccharide chains on glycoconjugates, either microbial or endogenous (host) sialidases (NanH, or N-acylneuraminate hydrolases) are needed to release free sugar, which is then transported by NanT in E. coli (15, 16, 26, 31). Once internalized, sialic acid is cleaved by an nanA-encoded aldolase or lyase to yield the 6-carbon hexosamine, N-acetylmannosamine (ManNAc), and pyruvate, with the latter entering the tricarboxylic acid cycle or gluconeogenesis. ManNAc is converted to its 6-phosphate derivative by a specific kinase encoded by nanK and epimerized by NanE to yield N-acetylglucosamine 6-phosphate, which is converted to fructose 6-phosphate by products of the nag operon (15, 17, 31, 32). The functions of the coregulated yjhS, yjhB, yjhC, and yhcH gene products are unknown but are not required for growth on Neu5Ac (15). However, YjhA (NanC) is an outer membrane porin required for diffusion of Neu5Ac in the absence of the major porins (7), while YjhT (NanM) is a mutarotase that catalyzes the conversion of the alpha sialic acid isomer to the more thermodynamically stable beta form (21). Neither nanC nor nanM is required for growth on Neu5Ac (15), suggesting that yjhS, yjhBC, and yhcH are involved in reactions that convert alternative sialic acids to Neu5Ac (22, 23). YhcH was crystallized and has been suggested to be an isomerase or epimerase involved in processing N-glycolylneuraminic acid (Neu5Gc) (25), but deletion of yhcH did not affect growth on this sialic acid as a sole carbon source (16).Computer-assisted analysis indicated that YjhB is a permease similar to NanT (16) whereas YjhC is a likely oxidoreductase or dehydrogenase. Orthologs of yhcH, nanC, nanM, and yjhBC are found in most bacterial species with intact Neu5Ac utilization systems, while yjhS is confined to E. coli and shigellae, either as part of the chromosomes in these strains or integrated with phages or phage remnants. However, a significant match (E value = 0.0007) was found between YjhS and AxeA in Rhodopirellula baltica, where AxeA is an acetyl xylan esterase (11), suggesting YjhS might be a sialate esterase. We propose that YjhS should be designated NanS to indicate its direct participation in utilization of an alternative sialic acid.     

Diversity of Dissimilatory Sulfite Reductase Genes (dsrAB) in a Salt Marsh Impacted by Long-Term Acid Mine Drainage

Sulfate-reducing bacteria (SRB) play a major role in the coupled biogeochemical cycling of sulfur and chalcophilic metal(loid)s. By implication, they can exert a strong influence on the speciation and mobility of multiple metal(loid) contaminants. In this study, we combined DsrAB gene sequencing and sulfur isotopic profiling to identify the phylogeny and distribution of SRB and to assess their metabolic activity in salt marsh sediments exposed to acid mine drainage (AMD) for over 100 years. Recovered dsrAB sequences from three sites sampled along an AMD flow path indicated the dominance of a single Desulfovibrio species. Other major sequence clades were related most closely to Desulfosarcina, Desulfococcus, Desulfobulbus, and Desulfosporosinus species. The presence of metal sulfides with low δ³⁴S values relative to δ³⁴S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of ∼2), although possibly within less acidic microenvironments. Interestingly, δ³⁴S values for pore water sulfate were lower than those for sulfate delivered during tidal inundation of marsh sediments. 16S rRNA gene sequence data from sediments and sulfur isotope data confirmed that sulfur-oxidizing bacteria drove the reoxidation of biogenic sulfide coupled to oxygen or nitrate reduction over a timescale of hours. Collectively, these findings imply a highly dynamic microbially mediated cycling of sulfate and sulfide, and thus the speciation and mobility of chalcophilic contaminant metal(loid)s, in AMD-impacted marsh sediments.Salt marshes exhibit high primary production rates (1, 101) and form biogeochemical “transition zones” for nutrient production, transport, and cycling between terrestrial and coastal marine environments (41, 66, 100). These zones also serve to reduce the flux of potentially toxic metals in contaminated groundwater to estuaries (12, 99, 106). Both functions depend strongly on microbial activity, especially that of sulfate-reducing bacteria (SRB) (42, 62, 67). SRB recycle much of the sedimentary organic carbon pool in marsh sediments (42-44) and indirectly inhibit production of the greenhouse gas methane (37, 71). They can restrict the mobility of dissolved contaminant metals by inducing precipitation of poorly soluble metal sulfides, and studies have examined their use in constructed wetlands to bioremediate acid mine drainage (AMD) and other metalliferous waste streams (11, 35, 40, 46, 50, 76, 90, 94, 104). However, the high acidity and metal concentrations inherent to AMD can inhibit SRB growth (15, 88, 98), and preferential growth of iron- and sulfur-oxidizing bacteria over SRB has been observed in some treatment wetlands (39).For natural salt marshes, 16S ribosomal nucleic acid- and phospholipid fatty acid (PLFA)-based analyses have shown that SRB commonly comprise a significant fraction of the microbial community (13, 24, 31, 34, 51, 58). Studies of salt marsh dissimilatory sulfite reductase genes (dsrAB), a highly conserved functional phylogenetic marker of prokaryotic sulfate reducers (49, 57, 102, 103, 107), have revealed both novel and deeply branching clades (3). Studies of mining-impacted sites at pH 2.0 to 7.8 (5, 7, 39, 70, 72, 77, 84), of soils and geothermal settings at a pH of ∼4 (55, 68), of metal-contaminated estuaries at pH 6.8 to 7.2 (65), and of hypersaline lakes at pH 7.5 (56) further outline the distribution and tolerance of specific groups and species of SRB under geochemically stringent conditions. Other findings point toward the existence of deltaproteobacteria in environments at a pH of ∼1 (10), although it is unknown if these include SRB. SRB diversity in salt marshes under long-term contamination by AMD has not been well investigated. Such studies may provide useful information for bioremediation projects in estuarine environments, as well as general insights into relationships between SRB physiology and the geochemistry of AMD.We studied the diversity of SRB, based on phylogenetic analysis of recovered DsrAB gene sequences (∼1.9 kb), in natural salt marsh sediments of the San Francisco Bay impacted by AMD for over 100 years. Sulfur isotope ratio and concentration measurements of pore water sulfate and metal sulfide minerals provided information about the spatial and temporal extent of active bacterial sulfate reduction (BSR) in sediment cores taken from specific sites along an AMD flow path. Collectively, the results revealed a tidal marsh system characterized by rapidly cycling bacterial sulfate reduction and sulfide reoxidation associated with oscillating tidal inundation and groundwater infiltration.     

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.     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.