AiiM,a Novel Class of N-Acylhomoserine Lactonase from the Leaf-Associated Bacterium Microbacterium testaceum

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signal molecules by many Gram-negative bacteria. We have reported that Microbacterium testaceum StLB037, which was isolated from the leaf surface of potato, has AHL-degrading activity. In this study, we cloned the aiiM gene from the genomic library of StLB037, which has AHL-degrading activity and shows high homology with the α/β hydrolase fold family from Actinobacteria. Purified AiiM as a maltose binding fusion protein showed high degrading activity of AHLs with both short- and long-chain AHLs with or without substitution at carbon 3. High-performance liquid chromatography analysis revealed that AiiM works as an AHL lactonase that catalyzes AHL ring opening by hydrolyzing lactones. In addition, expression of AiiM in the plant pathogen Pectobacterium carotovorum subsp. carotovorum reduced pectinase activity markedly and attenuated soft rot symptoms on potato slices. In conclusion, this study indicated that AiiM might be effective in quenching quorum sensing of P. carotovorum subsp. carotovorum.Quorum sensing is a cell-cell communication mechanism that depends on cell population density in bacteria (3, 7). In many Gram-negative bacteria, several kinds of N-acyl-l-homoserine lactones (AHLs) have been identified as signal compounds involved in this mechanism, and these are termed autoinducers (3, 7). AHL-mediated quorum sensing regulates the expression of many genes, including those responsible for bioluminescence, the production of pigments and antibiotics, and other processes (7). Many Gram-negative plant pathogens produce AHLs and regulate their virulence by AHL-mediated quorum sensing (31). For instance, Pectobacterium carotovorum subsp. carotovorum (formerly Erwinia carotovora), which causes soft rot diseases in many plant species, induces the production of various exoenzymes and plant tissue maceration by AHLs (1). Pantoea stewartii and Pantoea ananatis produce AHLs and regulate exopolysaccharide biosynthesis and the infection of plants (15, 32). In general, AHL-negative mutants show defects in pathogenicity, so it is expected that disrupting or manipulating quorum-sensing signals could inhibit the expression of virulence and infection of host cells.Recently, many AHL-degrading genes have been cloned and characterized from various bacteria. Genes encoding AHL lactonase, which catalyzes AHL ring opening by hydrolyzing lactones, have been cloned from Bacillus sp., Arthrobacter sp., Agrobacterium tumefaciens, and Rhodococcus erythropolis (5, 23, 30, 34). Genes encoding AHL acylase, which hydrolyze the amide bond of AHL, have been cloned from Ralstonia sp., Anabaena sp., Streptomyces sp., Shewanella sp., and Pseudomonas aeruginosa (11, 12, 16, 22, 25). Human and murine paraoxonase degrades AHL by hydrolyzing its lactone ring (21). Novel AHL lactonase genes have been isolated from a metagenomic library which was constructed from environmental soil samples (24, 27). AHL-degrading genes have also been utilized in the biocontrol of plant diseases. Expression of aiiA in transformed P. carotovorum subsp. carotovorum significantly attenuates pathogenicity on some crops (5). Transgenic plants expressing AHL lactonase exhibited significantly enhanced resistance to the infection of P. carotovorum subsp. carotovorum (4).We have reported the isolation of AHL-degrading Microbacterium testaceum StLB037 from the leaf surface of potato (Solanum tuberosum) (17). In coinfections, we found that StLB037 interrupted quorum-sensing-dependent bacterial infection by the plant pathogen P. carotovorum subsp. carotovorum. In this study, we report the cloning and characterization of a novel AHL lactonase gene (aiiM) from the chromosome of StLB037. In addition, we evaluated the potential use of heterologous aiiM gene expression in quenching quorum sensing in the plant pathogen P. carotovorum subsp. carotovorum.     

The cin and rai Quorum-Sensing Regulatory Systems in Rhizobium leguminosarum Are Coordinated by ExpR and CinS,a Small Regulatory Protein Coexpressed with CinI

To understand how the Rhizobium leguminosarum raiI-raiR quorum-sensing system is regulated, we identified mutants with decreased levels of RaiI-made N-acyl homoserine lactones (AHLs). A LuxR-type regulator, ExpR, is required for raiR expression, and RaiR is required to induce raiI. Since raiR (and raiI) expression is also reduced in cinI and cinR quorum-sensing mutants, we thought CinI-made AHLs may activate ExpR to induce raiR. However, added CinI-made AHLs did not induce raiR expression in a cinI mutant. The reduced raiR expression in cinI and cinR mutants was due to lack of expression of cinS immediately downstream of cinI. cinS encodes a 67-residue protein, translationally coupled to CinI, and cinS acts downstream of expR for raiR induction. Cloned cinS in R. leguminosarum caused an unusual collapse of colony structure, and this was delayed by mutation of expR. The phenotype looked like a loss of exopolysaccharide (EPS) integrity; mutations in cinI, cinR, cinS, and expR all reduced expression of plyB, encoding an EPS glycanase, and mutation of plyB abolished the effect of cloned cinS on colony morphology. We conclude that CinS and ExpR act to increase PlyB levels, thereby influencing the bacterial surface. CinS is conserved in other rhizobia, including Rhizobium etli; the previously observed effect of cinI and cinR mutations decreasing swarming in that strain is primarily due to a lack of CinS rather than a lack of CinI-made AHL. We conclude that CinS mediates quorum-sensing regulation because it is coregulated with an AHL synthase and demonstrate that its regulatory effects can occur in the absence of AHLs.Production of N-acyl homoserine lactones (AHLs) is common to many plant-associated bacteria (7), in which it is usually associated with population density-dependent regulation of genes affecting adaptive responses (49). Within the family Rhizobiaceae, population density-regulated gene expression (quorum sensing) mediated via AHLs has been identified in several agrobacteria and rhizobia (13, 51). In Agrobacterium spp., quorum-sensing regulation was initially identified as a mechanism of regulating plasmid transfer. As the bacterial population density increases, plasmid transfer genes are induced by TraR in response to AHLs made by TraI (55). In several rhizobia, traI-like AHL synthase genes are also in an operon along with plasmid transfer genes (13).There are other quorum-sensing loci in different strains of rhizobia. In Sinorhizobium meliloti strain Rm1021, AHLs produced by SinI activate SinR and ExpR, LuxR-type regulators, to induce several genes, including those determining the production of an exopolysaccharide, exopolysaccharide II (EPS-II) (17, 23, 24, 35), that plays an important role in the symbiosis. In S. meliloti, two LuxR-type regulators, VisN and VisR, are involved in chemotaxis and motility (24, 44). Rhizobium etli has multiple AHL synthase genes (9, 39), but the functions of many of the regulated genes remain to be established. The cinR and cinI genes are required for normal symbiotic nitrogen fixation and swarming in R. etli (5, 9, 11) and for normal levels of expression of raiI, which encodes another AHL synthase. The expression of raiI in R. etli is regulated by RaiR (39).Analysis of AHLs produced by strain A34 of Rhizobium leguminosarum bv. viciae led to the characterization of four LuxI-type AHL synthases (RhiI, CinI, RaiI, and TraI) and five LuxR-type regulators (RhiR, CinR, RaiR, TraR, and BisR) (8, 31, 50, 53). In this strain, the cinI and cinR genes are chromosomally located; CinI produces N-(3-hydroxy-7-cis-tetradecenoyl)-l-homoserine lactone (3-OH-C_14:1-HSL) (20, 31), CinR induces cinI expression in response to this AHL (31), and this appears to be associated with adaptation to starvation and salt stress (47). Mutation of cinI or cinR affects the expression of the other three AHL synthase genes in R. leguminosarum bv. viciae strain A34. Thus, in a cinI mutant, the expression of raiI is reduced, resulting in very low levels of 3-OH-C₈-HSL, the major AHL made by RaiI (53). Similarly, the expression levels of the traI and rhiI genes on the symbiotic plasmid pRL1JI are reduced in cinI and cinR mutants (31). RhiI-made AHLs activate RhiR to induce the expression of the rhiABC operon in R. leguminosarum bv. viciae (38), enhancing the interaction with the legume host (8).The cinI and cinR quorum-sensing genes control induction of the traI and traR quorum-sensing regulons via CinI-made 3-OH-C_14:1-HSL, which activates BisR (another LuxR-type regulator) to induce traR and hence traI (12). However, the mechanism by which cinI and/or cinR control raiI and raiR expression has not been established. In this work we demonstrate that raiI and raiR expression requires both expR and a small gene (cinS) cotranscribed with cinI. CinS also regulates the expression of plyB encoding an extracellular glycanase and is required for swarming of R. etli.     

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.     

Social Interactions and Distribution of Bacillus subtilis Pherotypes at Microscale

Bacillus subtilis strains communicate through the comQXPA quorum sensing (QS) system, which regulates genes expressed during early stationary phase. A high polymorphism of comQXP′ loci was found in closely related strains isolated from desert soil samples separated by distances ranging from meters to kilometers. The observed polymorphism comprised four communication groups (pherotypes), such that strains belonging to the same pherotype exchanged information efficiently but strains from different pherotypes failed to communicate. To determine whether the same level of polymorphism in the comQXP′ QS system could be detected at microscale, B. subtilis isolates were obtained from two separate 1-cm³ soil samples, which were progressively divided into smaller sections. Cross-activation studies using pherotype-responsive reporter strains indicated the same number of communication pherotypes at microscale as previously determined at macroscale. Sequencing of the housekeeping gene gyrA and the QS comQ gene confirmed different evolutionary rates of these genes. Furthermore, an asymmetric communication response was detected inside the two pherotype clusters, suggesting continuous evolution of the QS system and possible development of new languages. To our knowledge, this is the first microscale study demonstrating the presence of different QS languages among isolates of one species, and the implications of this microscale diversity for microbial interactions are discussed.Quorum sensing (QS), a widespread phenomenon in the bacterial world, controls a wide range of cell density-dependent behaviors. Bacillus subtilis uses QS to control production of antimicrobial peptides, bacteriocins, and antibiotics (20) but also to alternate between two cell types during stationary phase: competent cells, able to take in DNA from the environment, and dormant spores, able to survive harsh environmental conditions (9, 12, 24). Development of genetic competence in B. subtilis is controlled by a QS system encoded by the comQXPA operon (2, 53, 54). This involves the ComX pheromone that accumulates during exponential growth (25, 46, 47) and is initially synthesized as a 55-residue protein that is processed, modified, and released into the extracellular medium as a 5- to 10-amino-acid peptide. The isoprenoidal modification on the tryptophan residue of this peptide is catalyzed by the ComQ protein (2, 25, 34, 35, 42, 52). Upon reaching the threshold concentration, processed and modified ComX binds to the membrane-associated, histidine protein kinase ComP and triggers the QS response, linking autophosphorylation of ComP and transfer of phosphate to the response regulator ComA (59). The level of phosphorylated ComA is also controlled by dephosphorylation, which is dependent on a separate QS system involving competence sporulation factor (CSF) and the RapC phosphatase (3, 59). Phosphorylated ComA directly controls expression of various genes (6, 33), including the srfAB operon that contains the comS gene (15, 41), required for development of competence (55).Previous studies of environmental B. subtilis strains indicate a high polymorphism (approximately 56% identity at the nucleotide level) in the QS locus, which is restricted to comQ, comX, and the N-terminal region of the comP gene. Sequences surrounding this locus, downstream gene comA, a C-terminal region of comP, and the upstream degQ gene, are highly conserved (2, 53, 54). Sequence analysis of the comQXP loci of 13 strains indicated clustering into four distinct similarity groups (2). These groups were congruent for comQ, comX, and the N-terminal region of comP, indicating coevolution of the three genes. In addition, the similarity groups correlated with four pherotypes, able to communicate efficiently within but not between groups. Similar variation has been reported for the agr QS system in staphylococci (19, 56) and in the competence QS system of Streptococcus pneumoniae (17, 19, 37, 38, 60).B. subtilis is often referred to as a soil-dwelling organism, its spores persisting in soil until encountering conditions suitable for germination and growth (10). The basic structural unit of soil ecosystems is the soil aggregate, in which biogeochemical processes occur at scales relevant to microorganisms. Approximately 50% of the volume of a soil aggregate represents open pores, while the remainder consists of mineral particles (sand, silt, and clay) held together by organic material (48), with which B. subtilis may be preferentially associated (16, 43). Soil aggregates can be classified as macroaggregates (diameter, >250 μm) and microaggregates (diameter, 2 to 250 μm) (39), but little is known about the distribution of bacteria within aggregates. Structural organization of the soil creates a mosaic of microenvironments, within which water movement and diffusion of nutrients and other molecules play key roles in functioning of the soil microbiota (7, 13, 39). These roles may vary with the scale at which they operate. Tisdall and Oades (51) suggest that scales at which microorganisms are important in the soil aggregation process range between 2 and 2,000 μm, depending on the specific system being investigated (13). Although the microscale distribution of microorganisms and their associated functions have rarely been studied, it is becoming recognized that greater knowledge of spatial organization at the scale of a soil aggregate (microscale) is essential for a better understanding of soil ecosystem function and of the mechanisms that generate and maintain diversity, including speciation, extinction, dispersal, and interactions within and between species (7, 13, 26).The aim of this study was to assess the potential role of QS in generating and maintaining microscale diversity within the soil. This was achieved by determining the genomic and functional diversification of the B. subtilis QS system with regard to geographical distance and ecological characteristics. Isolates were obtained from two 1-cm³ sandy, riverbank soil samples separated by approximately 5 m, allowing assessment of macroscale diversity. In addition, each riverbank soil sample was treated as a separate macroaggregate that was progressively sectioned to obtain subsamples of different sizes, allowing assessment of microscale diversity. The riverbank soil B. subtilis isolates were compared with Bacillus isolates previously obtained from desert soil samples separated by distances of meters to kilometers (2, 40), representing macroscale distribution. The Bacillus isolates were used to (i) correlate geographical distance (microscale/macroscale) with genomic distance of the QS comQ gene and the housekeeping gyrA gene, (ii) investigate and compare the specificity of the QS response of microscale and macroscale isolates, and (iii) explore dominance of pherotypes inside soil aggregates. To our knowledge, this is the first investigation of a QS system that addresses the genomic and functional diversification of bacterial populations at microscale.     

Inhibition of the Early Stage of Salmonella enterica Serovar Enteritidis Biofilm Development on Stainless Steel by Cell-Free Supernatant of a Hafnia alvei Culture

Compounds present in Hafnia alvei cell-free culture supernatant cumulatively negatively influence the early stage of biofilm development by Salmonella enterica serovar Enteritidis on stainless steel while they also reduce the overall metabolic activity of S. Enteritidis planktonic cells. Although acylhomoserine lactones (AHLs) were detected among these compounds, the use of several synthetic AHLs was not able to affect the initial stage of biofilm formation by this pathogen.Biofilms are groups of bacteria encased in a self-produced extracellular matrix (5, 6). Biofilms formed on stainless steel (SS) surfaces in food-processing areas are of great importance since they may lead to food spoilage and transmission of diseases (2, 16). This sessile mode of life allows bacteria to enjoy a number of advantages, such as increased resistance to antimicrobial agents (9, 12). Notably, it is widely accepted that bacteria (both planktonic and biofilm cells) communicate by releasing and sensing signaling compounds in a process commonly known as quorum sensing (13, 18, 24).Salmonella enterica serovar Enteritidis is one of the most important bacterial pathogens worldwide (7, 17). Hafnia alvei are frequent psychrotrophic members of the Enterobacteriaceae community in meat products, playing a role in their spoilage, while they have been shown to be capable of producing signaling compounds (3). In this study, in order to determine any possible influence of compounds produced by H. alvei on the biofilm-forming ability of S. Enteritidis, the latter was left to develop biofilm on SS surfaces in the presence of conditioned medium obtained after the growth of the former. Biofilm formation was assessed directly by detaching cells and enumerating them and, also, indirectly by automated conductance measurements.     

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).     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

A Bacillus anthracis S-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC

The sequestration of iron by mammalian hosts represents a significant obstacle to the establishment of a bacterial infection. In response, pathogenic bacteria have evolved mechanisms to acquire iron from host heme. Bacillus anthracis, the causative agent of anthrax, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, including B. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Here, we present evidence that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation of B. anthracis to changing iron reservoirs during an infection. Understanding the mechanism of heme uptake in pathogenic bacteria is important for the development of novel therapeutics to prevent and treat bacterial infections.Pathogenic bacteria need to acquire iron to survive in mammalian hosts (12). However, the host sequesters most iron in the porphyrin heme, and heme itself is often bound to proteins such as hemoglobin (14, 28, 85). Circulating hemoglobin can serve as a source of heme-iron for replicating bacteria in infected hosts, but the precise mechanisms of heme extraction, transport, and assimilation remain unclear (25, 46, 79, 86). An understanding of how bacterial pathogens import heme will lead to the development of new anti-infectives that inhibit heme uptake, thereby preventing or treating infections caused by these bacteria (47, 68).The mechanisms of transport of biological molecules into a bacterial cell are influenced by the compositional, structural, and topological makeup of the cell envelope. Gram-negative bacteria utilize specific proteins to transport heme through the outer membrane, periplasm, and inner membrane (83, 84). Instead of an outer membrane and periplasm, Gram-positive bacteria contain a thick cell wall (59, 60). Proteins covalently anchored to the cell wall provide a functional link between extracellular heme reservoirs and intracellular iron utilization pathways (46). In addition, several Gram-positive and Gram-negative bacterial genera also contain an outermost structure termed the S (surface)-layer (75). The S-layer is a crystalline array of protein that surrounds the bacterial cell and may serve a multitude of functions, including maintenance of cell architecture and protection from host immune components (6, 7, 18, 19, 56). In bacterial pathogens that manifest an S-layer, the “force field” function of this structure raises questions concerning how small molecules such as heme can be successfully passed from the extracellular milieu to cell wall proteins for delivery into the cell cytoplasm.Bacillus anthracis is a Gram-positive, spore-forming bacterium that is the etiological agent of anthrax disease (30, 33). The life cycle of B. anthracis begins after a phagocytosed spore germinates into a vegetative cell inside a mammalian host (2, 40, 69, 78). Virulence determinants produced by the vegetative cells facilitate bacterial growth, dissemination to major organ systems, and eventually host death (76-78). The release of aerosolized spores into areas with large concentrations of people is a serious public health concern (30).Heme acquisition in B. anthracis is mediated by the action of IsdX1 and IsdX2, two extracellular hemophores that extract heme from host hemoglobin and deliver the iron-porphyrin to cell wall-localized IsdC (21, 45). Both IsdX1 and IsdX2 harbor near iron transporter domains (NEATs), a conserved protein module found in Gram-positive bacteria that mediates heme uptake from hemoglobin and contributes to bacterial pathogenesis upon infection (3, 8, 21, 31, 44, 46, 49, 50, 67, 81, 86). Hypothesizing that B. anthracis may contain additional mechanisms for heme transport, we provide evidence that B. anthracis S-layer protein K (BslK), an S-layer homology (SLH) and NEAT protein (32, 43), is surface localized and binds and transfers heme to IsdC in a rapid, contact-dependent manner. These results suggest that the Isd system is not a self-contained conduit for heme trafficking and imply that there is functional cross talk between differentially localized NEAT proteins to promote heme uptake during infection.