Central Role of the Early Colonizer Veillonella sp. in Establishing Multispecies Biofilm Communities with Initial,Middle, and Late Colonizers of Enamel

Human dental biofilm communities comprise several species, which can interact cooperatively or competitively. Bacterial interactions influence biofilm formation, metabolic changes, and physiological function of the community. Lactic acid, a common metabolite of oral bacteria, was measured in the flow cell effluent of one-, two- and three-species communities growing on saliva as the sole nutritional source. We investigated single-species and multispecies colonization by using known initial, early, middle, and late colonizers of enamel. Fluorescent-antibody staining and image analysis were used to quantify the biomass in saliva-fed flow cells. Of six species tested, only the initial colonizer Actinomyces oris exhibited significant growth. The initial colonizer Streptococcus oralis produced lactic acid but showed no significant growth. The early colonizer Veillonella sp. utilized lactic acid in two- and three-species biofilm communities. The biovolumes of all two-species biofilms increased when Veillonella sp. was present as one of the partners, indicating that this early colonizer promotes mutualistic community development. All three-species combinations exhibited enhanced growth except one, i.e., A. oris, Veillonella sp., and the middle colonizer Porphyromonas gingivalis, indicating specificity among three-species communities. Further specificity was seen when Fusobacterium nucleatum (a middle colonizer), Aggregatibacter actinomycetemcomitans (a late colonizer), and P. gingivalis did not grow with S. oralis in two-species biofilms, but inclusion of Veillonella sp. resulted in growth of all three-species combinations. We propose that commensal veillonellae use lactic acid for growth in saliva and that they communicate metabolically with initial, early, middle, and late colonizers to establish multispecies communities on enamel.The human oral cavity contains a widely diverse community of resident bacteria composed of several hundred species (1, 18). They organize into multispecies communities through a recurrent sequence of colonization that occurs after each oral hygiene treatment; for example, dental plaque development on enamel starts with the initial colonizers streptococci and actinomyces (7, 15), which are followed by early-colonizing veillonellae (7, 11, 14), middle-colonizing porphyromonads (7) and fusobacteria (7, 10, 11), and late-colonizing aggregatibacters (9).During the initial stage of biofilm formation, streptococci and actinomyces bind to host-derived receptors in the salivary pellicle coating of enamel. In turn, other species bind to already-adherent cells, a process called coadhesion (2). This process and coaggregation (10), defined as specific cell-to-cell recognition between genetically distinct cells, as well as growth of adherent cells contribute to dental plaque development. While it is known that pure cultures of oral bacteria metabolize dietary sugars to lactic acid, little is known about the importance of lactic acid to community growth on saliva as a sole nutrient source. Most pure cultures and many combinations of species are unable to grow on whole saliva, which is a complex nutritional source. Growth might, in fact, require spatial organization and mutualistic interactions among selected species that collectively possess a combination of metabolic properties that are capable of converting latent nutrition into usable nutrition. In succession, groups of other selected species with other combined metabolic capabilities can further process this complex nutritional source, with a resultant assembling and disassembling of constantly changing oral biofilm communities.Streptococci make up 60 to 90% of the supragingival plaque biomass in the first 24 h of colonization (12, 15). They catabolize carbohydrates to short-chain organic acids, such as lactic acid and pyruvic acid (4). Veillonellae constitute as much as 5% of the initial plaque biomass but are unable to catabolize sugars. They rely on the fermentation of organic acids such as lactic acid (6) and thus set up a convenient metabolic food chain in dental plaque.In vivo studies using gnotobiotic rats demonstrated that veillonellae were unable to establish monoinfections. Yet when a strain of Veillonella was inoculated into rats already monoinfected with a strain of Streptococcus mutans that coaggregates with that Veillonella strain, the number of veillonellae on the teeth of the coinfected animals was 1,000-fold higher than the number when a noncoaggregating Veillonella strain was used (13). Also, in gnotobiotic rats, lower caries and plaque scores were obtained for two-species biofilms than for single-species colonization by streptococci, and inclusion of veillonellae reduced caries activity and demineralization of the enamel by streptococci (13). Streptococcus-Veillonella communities containing coaggregation partners were micromanipulated from 8-h human dental plaque, providing additional evidence of the close association of these two species in vivo (3). Further, Veillonella spp. are juxtaposed with coaggregation receptor polysaccharide-bearing streptococci in early communities in vivo, and a rapid succession of veillonella phylotypes occurs in these communities (16). These reports offer broad-based evidence that veillonellae and streptococci are linked in oral biofilms.The focus of the current investigation was to explore Veillonella-based mixed-species communities in saliva-fed flow cells. The concentration of lactic acid in the effluent of flow cells containing biofilm communities was determined. We hypothesize that spatiotemporal metabolic interactions and coaggregation of Veillonella sp. with Streptococcus oralis and early, middle, and late colonizers allow these organisms to form three-species biofilm communities. We show high specificity of community partnerships among the six species examined, suggesting that successions of species in naturally recurring dental plaque in vivo are centered on metabolic and physical interactions of the community participants which support the nonrandom sequential appearance of species in the development of oral biofilms.     

The Native 67-Kilodalton Minor Fimbria of Porphyromonas gingivalis Is a Novel Glycoprotein with DC-SIGN-Targeting Motifs

We recently reported that the oral mucosal pathogen Porphyromonas gingivalis, through its 67-kDa Mfa1 (minor) fimbria, targets the C-type lectin receptor DC-SIGN for invasion and persistence within human monocyte-derived dendritic cells (DCs). The DCs respond by inducing an immunosuppressive and Th2-biased CD4⁺ T-cell response. We have now purified the native minor fimbria by ion-exchange chromatography and sequenced the fimbria by tandem mass spectrometry (MS/MS), confirming its identity and revealing two putative N-glycosylation motifs as well as numerous putative O-glycosylation sites. We further show that the minor fimbria is glycosylated by ProQ staining and that glycosylation is partially removed by treatment with β(1-4)-galactosidase, but not by classic N- and O-linked deglycosidases. Further monosaccharide analysis by gas chromatography-mass spectrometry (GC-MS) confirmed that the minor fimbria contains the DC-SIGN-targeting carbohydrates fucose (1.35 nmol/mg), mannose (2.68 nmol/mg), N-acetylglucosamine (2.27 nmol/mg), and N-acetylgalactosamine (0.652 nmol/mg). Analysis by transmission electron microscopy revealed that the minor fimbria forms fibers approximately 200 nm in length that could be involved in targeting or cross-linking DC-SIGN. These findings shed further light on molecular mechanisms of invasion and immunosuppression by this unique mucosal pathogen.Porphyromonas gingivalis is one of several mucosal pathogens that have been implicated in chronic periodontitis (CP), a common oral disease that may affect 40 to 60% of the U.S. population (7). P. gingivalis utilizes a myriad of virulence factors that contribute to chronic periodontitis. Among these are a polysaccharide capsule, fimbriae, proteases for opsonins C3 and IgG, gingipains (21, 30, 43, 52), bacterial lipopolysaccharides (LPS) (22, 44), and toxins and hemagglutinins (10, 25).The fimbriae of P. gingivalis play a crucial role in adhesion to and invasion of host cells. We have shown that optimum entry of P. gingivalis into human dendritic cells (DCs) requires the presence of two fimbriae, termed the major and minor fimbriae. The major fimbria is composed of a 41-kDa protein termed fimbrillin, encoded by the fimA gene (65). Much less is known about the minor fimbria, the focus of this paper. The minor fimbria is comprised of a 67-kDa protein (19) that is encoded by the mfa1 gene. The major and minor fimbriae are antigenically distinct, and they also differ based on amino acid composition and size (5, 19). Very little is understood about the formation and secretion of the minor fimbriae and about possible posttranslational modifications of these fimbriae. Formation and secretion of the major fimbriae is a complex reaction consisting of numerous steps required for transfer of prefimbrillin proteins from the cytoplasm to the periplasm, cleavage of the N-terminal signal peptide (24, 50), transport of prefimbrillin to the outer face of the outer membrane, and assembly into fimbria structures (23, 24, 34).Deciphering the cellular receptors for the fimbriae is an active area of research. Evidence suggests that the cellular targets of the major fimbriae are the β-1 integrins (CD29) (32, 66). Others have proposed a role for β-2 integrins (CD18) (17, 18, 55) in the cellular response to major fimbriae. In contrast, little is known of the cellular receptors for the minor fimbriae. Lamont et al. in 2002 showed that the minor fimbria of P. gingivalis intimately interacts with the SspB protein of Streptococcus gordonii (26). This interaction might aid in P. gingivalis colonization of plaque biofilm before it invades gingival tissue (26, 41). We recently showed that the minor fimbria targets DC-SIGN on DCs for entry into DCs and that this targeting has the immunological consequence of dampening the immune response (68).DC-SIGN is a type II membrane protein on DCs in which the extracellular domain consists of a stalk that promotes tetramerization (13). DC-SIGN contains a C-terminal carbohydrate-recognizing domain (CRD) that belongs to the C-type lectin superfamily (13). Early studies by Feinberg et al. in 2001 showed that the DC-SIGN CRD preferentially binds to the high-mannose N-linked oligosaccharides GlcNAc (N-acetylglucosamine) and Manα1-3[Manα1-6] Man (mannose) (13). Furthermore, Appelmelk et al. showed that DC-SIGN also binds to fucose-containing Lewis blood antigens (4). Guo et al. utilized an extensive glycan array and showed that DC-SIGN will bind high-mannose-containing glycans or glycans that contain terminal fucose residues (16). Previous studies showed that DC-SIGN on DCs is used by microorganisms such as Neisseria gonorrhoeae, Mycobacterium tuberculosis, Mycobacterium leprae, HIV, and Helicobacter pylori for entry into DCs and induction of immunosuppression (4, 27, 42, 51, 69). Like P. gingivalis, many of these pathogens can induce chronic life-long infections.Our previously published work established that the minor fimbria is necessary for targeting DC-SIGN, resulting in entry of P. gingivalis into DCs (68). We were able to abrogate minor fimbria-mediated DC-SIGN ligation by using DC-SIGN-blocking agents or agonists, including fucose, mannose, and mannan (68). Additionally, we described that the minor fimbria is able to induce immunosuppression of DCs via its interaction with DC-SIGN, which was blocked by sugars (68). Further, we demonstrated that minor fimbriated strains of P. gingivalis inhibited DC maturation and suppressed proinflammatory cytokine secretion (68). Moreover, DCs that were pulsed with minor fimbriated strains of P. gingivalis and then cocultured with autologous T cells shifted the T-cell effector phenotype to a Th2 effector phenotype, as evidenced by high interleukin-4 (IL-4) production (68).Our previous results, described above, suggested that the minor fimbria-DC-SIGN interaction was mediated by glycosylated proteins. We therefore set out to identify the carbohydrate moieties on the minor fimbria that could account for its DC-SIGN-targeting function. The intact native minor fimbria was purified and analyzed for glycosylation and for the presence of relevant monosaccharides. We show here by a combination of ProQ gel staining and gas chromatography-mass spectrometry (GC-MS) analysis that the minor fimbria is glycosylated and expresses the DC-SIGN ligands fucose, mannose, GlcNAc, and GalNAc. Use of classic N- and O-linked deglycosidases on the native minor fimbria revealed a novel glycoprotein structure. Overall, these results indicate that the minor fimbria is glycosylated with DC-SIGN-binding motifs that likely account for the reported ability of P. gingivalis to bind to and invade DCs, resulting in an immunosuppressive DC response.     

Characterization of Hydrogen Peroxide-Induced DNA Release by Streptococcus sanguinis and Streptococcus gordonii

Extracellular DNA (eDNA) is produced by several bacterial species and appears to contribute to biofilm development and cell-cell adhesion. We present data showing that the oral commensals Streptococcus sanguinis and Streptococcus gordonii release DNA in a process induced by pyruvate oxidase-dependent production of hydrogen peroxide (H₂O₂). Surprisingly, S. sanguinis and S. gordonii cell integrity appears unaffected by conditions that cause autolysis in other eDNA-producing bacteria. Exogenous H₂O₂ causes release of DNA from S. sanguinis and S. gordonii but does not result in obvious lysis of cells. Under DNA-releasing conditions, cell walls appear functionally intact and ribosomes are retained over time. During DNA release, intracellular RNA and ATP are not coreleased. Hence, the release mechanism appears to be highly specific for DNA. Release of DNA without detectable autolysis is suggested to be an adaptation to the competitive oral biofilm environment, where autolysis could create open spaces for competitors to invade. Since eDNA promotes cell-to-cell adhesion, release appears to support oral biofilm formation and facilitates exchange of genetic material among competent strains.The release of bacterial DNA into the environment is of recent interest since this polymer is now recognized to stabilize cell-to-cell adherence and biofilm architecture (1, 35, 37). Treatment of extracellular DNA (eDNA) with DNase results in reduced intercellular stickiness, consistent with an adhesive function for eDNA. Furthermore, eDNA from Neisseria meningitis appears to have sufficient structural integrity to transform competent strains (11), indicating chromosomal origin. Since the abundance of eDNA is influenced by growth conditions, DNA release can also be regulated (40).DNA release is typically a consequence of cell lysis. Linked to DNA release, genetic transformation is the natural ability of competent bacterial species to take up DNA from the environment (13, 34, 42). During competence development, Streptococcus pneumoniae DNA is released by lysis of a subpopulation of cells (30, 42). Cell lysis and DNA release are controlled in a cell density-dependent signal transduction process. The S. pneumoniae comX regulon, carrying late competence genes, also includes the murein hydrolase genes lytA and cbpD (19, 42). Murein hydrolases digest structural components of the peptidoglycan, contributing to remodeling, recycling, and daughter cell separation. Furthermore, murein hydrolases trigger autolytic cell wall digestion, leading to release of DNA and other cellular content into the environment (36). The autolysis of bacterial cells as part of a regulated death program seems to be an important source for eDNA in diverse species, including Staphylococcus aureus (4, 36, 37), Staphylococcus epidermidis (35), Enterococcus faecalis (44), and Pseudomonas aeruginosa (1). In these species, the eDNA contributes to biofilm formation as a component of the extracellular biofilm matrix (35, 37, 44).Unlike for cell lysis-dependent release, the oral streptococci appear to induce eDNA release by a novel mechanism. In dual-species cultures, the oral commensals Streptococcus sanguinis and Streptococcus gordonii release eDNA in a manner dependent on pyruvate oxidase (Pox) generation of hydrogen peroxide (H₂O₂) under the control of ambient oxygen (23). In this report, we now provide direct evidence of selective H₂O₂-induced eDNA release by these oral commensal streptococci.     

Fusobacterium nucleatum ATCC 10953 Requires Actinomyces naeslundii ATCC 43146 for Growth on Saliva in a Three-Species Community That Includes Streptococcus oralis 34

Formation of dental plaque is a developmental process involving initial and late colonizing species that form polymicrobial communities. Fusobacteria are the most numerous gram-negative bacteria in dental plaque, but they become prevalent after the initial commensal colonizers, such as streptococci and actinomyces, have established communities. The unusual ability of these bacteria to coaggregate with commensals, as well as pathogenic late colonizers, has been proposed to facilitate colonization by the latter organisms. We investigated the integration of Fusobacterium nucleatum into multispecies communities by employing two in vitro models with saliva as the sole nutritional source. In flow cell biofilms, numbers of cells were quantified using fluorescently conjugated antibodies against each species, and static biofilms were analyzed by quantitative real-time PCR (q-PCR) using species-specific primers. Unable to grow as single-species biofilms, F. nucleatum grew in two-species biofilms with Actinomyces naeslundii but not with Streptococcus oralis. However, enhanced growth of fusobacteria was observed in three-species biofilms, indicating that there was multispecies cooperation. Importantly, these community dynamics yielded an 18-fold increase in the F. nucleatum biomass between 4 h and 18 h in the flow cell inoculated with three species. q-PCR analysis of static biofilms revealed that maximum growth of the three species occurred at 24 h to 36 h. Lower numbers of cells were observed at 48 h, suggesting that saliva could not support higher cell densities as the sole nutrient. Integration of F. nucleatum into multispecies commensal communities was evident from the interdigitation of fusobacteria in coaggregates with A. naeslundii and S. oralis and from the improved growth of fusobacteria, which was dependent on the presence of A. naeslundii.The human mouth contains microbiologically diverse communities. While collectively humans harbor more than 700 bacterial phylotypes, each individual is estimated to have fewer than 100 such phylotypes (1), and approximately 50% of human oral bacteria have yet to be cultivated. Although biofilm communities on tooth enamel are polymicrobial (3, 20), more than 60 to 90% of the bacteria found in initial plaque on saliva-coated tooth enamel are streptococci (6, 19). Other bacterial genera that are among the initial commensal colonizers include Actinomyces, Veillonella, and Neisseria (6, 16, 19), and these organisms contribute to the polymicrobial nature of initial plaque.The structure of a community is dependent upon the nature of the foundation. An integral feature of an oral bacterial biofilm foundation is the ability to coaggregate, which is defined as cell-cell recognition and binding between genetically distinct bacteria. After routine oral hygiene treatment, freshly cleaned tooth enamel is quickly coated with a salivary pellicle, which provides a set of receptor molecules recognized by primary colonizing bacteria, such as streptococci and actinomyces. Besides recognizing salivary receptors, these bacteria coaggregate and provide a foundation for the subsequent attachment and growth of other bacteria, such as veillonellae, that form close metabolic relationships with streptococci (12, 15). As initial colonizers develop into biofilm communities with anaerobic microenvironments, incorporation of the obligate anaerobic fusobacteria into these communities becomes possible. Fusobacteria as a group coaggregate with all other oral bacteria and have been suggested, therefore, to be a crucial link between primary colonizing species and later colonizing pathogens (13, 14). Thus, a foundation consisting of coaggregating streptococci, actinomyces, and veillonellae populates the tooth surface, and these organisms are recognized by fusobacteria, which colonize and become the dominant gram-negative bacterial species. The new foundation is a substratum containing fusobacterial surface receptors available for recognition by late colonizing pathogens. Supporting the crucial link is clinical evidence that fusobacteria appear in dental plaque after commensal species and before the pathogenic “red” complex consisting of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia (22, 23).Coaggregation partnerships are highly specific. A significant role for coaggregation in the formation of dental plaque biofilms and particularly in accretion of secondary colonizers to the pioneer species in plaque has been proposed (14) and has been demonstrated for the development of a spatially organized community (20). However, coaggregation may also provide some metabolic advantages (e.g., cross feeding and enzyme complementation) to neighboring cells by facilitating physical juxtaposition of partner cells, as has been shown for glucose metabolism of coaggregates of actinomyces and streptococci (7, 8). One aim of the present study was to examine the structures of two- and three-species communities composed of Actinomyces naeslundii, Streptococcus oralis, and Fusobacterium nucleatum in model biofilm systems. The first two species are initial colonizers and are considered commensals, whereas fusobacteria are secondary colonizers and are postulated to be a coaggregation bridge between initial and late colonizers (14). Our second aim was to investigate the integration and growth of fusobacteria in polymicrobial communities.A variety of experimental methods have been developed to study the formation of biofilms. Model systems often rely on the flow of nutrients over a surface on which bacteria are able to attach and grow. In the present study we used two distinct in vitro models, a saliva-fed flow cell and a polystyrene peg immersed in static saliva. Biofilm communities form naturally and are undisturbed (3, 20, 21). The spatial organization of a multispecies community resulting from colonization and growth is preserved and can be examined noninvasively by confocal laser scanning microscopy (CLSM). In the static system, the amount of each species in multispecies biofilms formed on polystyrene pegs can be measured by real-time quantitative PCR (q-PCR). We show here with both models that fusobacteria are unable to grow as single species, but they integrate into commensal streptococcus-actinomyces communities and grow. Integration and growth are required for fusobacteria to become crucial links between commensal communities and later colonizing pathogenic communities. In the three-species community studied here, A. naeslundii is required for F. nucleatum to integrate and grow.     

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).     

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.     

Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

An IcmF Family Protein,ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL,and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens

An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpL_M, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpL_M and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpL_M is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impL_M mutants with substitutions or deletions in the Walker A motif failed to complement the impL_M deletion mutant for Hcp secretion, which provided evidence that ImpL_M may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpL_M and another essential T6SS component, ImpK_L. Topology and biochemical fractionation analyses suggested that ImpK_L is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpL_M-ImpK_L interaction domains suggested that ImpL_M interacts with ImpK_L via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL_M interacts with ImpK_L, and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.Many pathogenic gram-negative bacteria employ protein secretion systems formed by macromolecular complexes to deliver proteins or protein-DNA complexes across the bacterial membrane. In addition to the general secretory (Sec) pathway (18, 52) and twin-arginine translocation (Tat) pathway (7, 34), which transport proteins across the inner membrane into the periplasm, at least six distinct protein secretion systems occur in gram-negative bacteria (28, 46, 66). These systems are able to secrete proteins from the cytoplasm or periplasm to the external environment or the host cell and include the well-documented type I to type V secretion systems (T1SS to T5SS) (10, 15, 23, 26, 30) and a recently discovered type VI secretion system (T6SS) (4, 8, 22, 41, 48, 49). These systems use ATPase or a proton motive force to energize assembly of the protein secretion machinery and/or substrate translocation (2, 6, 41, 44, 60).Agrobacterium tumefaciens is a soilborne pathogenic gram-negative bacterium that causes crown gall disease in a wide range of plants. Using an archetypal T4SS (9), A. tumefaciens translocates oncogenic transferred DNA and effector proteins to the host and ultimately integrates transferred DNA into the host genome. Because of its unique interkingdom DNA transfer, this bacterium has been extensively studied and used to transform foreign DNA into plants and fungi (11, 24, 40, 67). In addition to the T4SS, A. tumefaciens encodes several other secretion systems, including the Sec pathway, the Tat pathway, T1SS, T5SS, and the recently identified T6SS (72). T6SS is highly conserved and widely distributed in animal- and plant-associated Proteobacteria and plays an important role in the virulence of several human and animal pathogens (14, 19, 41, 48, 56, 63, 74). However, T6SS seems to play only a minor role or even a negative role in infection or virulence of the plant-associated pathogens or symbionts studied to date (5, 37-39, 72).T6SS was initially designated IAHP (IcmF-associated homologous protein) clusters (13). Before T6SS was documented by Pukatzki et al. in Vibrio cholerae (48), mutations in this gene cluster in the plant symbiont Rhizobium leguminosarum (5) and the fish pathogen Edwardsiella tarda (51) caused defects in protein secretion. In V. cholerae, T6SS was responsible for the loss of cytotoxicity for amoebae and for secretion of two proteins lacking a signal peptide, hemolysin-coregulated protein (Hcp) and valine-glycine repeat protein (VgrG). Secretion of Hcp is the hallmark of T6SS. Interestingly, mutation of hcp blocks the secretion of VgrG proteins (VgrG-1, VgrG-2, and VgrG-3), and, conversely, vgrG-1 and vgrG-2 are both required for secretion of the Hcp and VgrG proteins from V. cholerae (47, 48). Similarly, a requirement of Hcp for VgrG secretion and a requirement of VgrG for Hcp secretion have also been shown for E. tarda (74). Because Hcp forms a hexameric ring (41) stacked in a tube-like structure in vitro (3, 35) and VgrG has a predicted trimeric phage tail spike-like structure similar to that of the T4 phage gp5-gp27 complex (47), Hcp and VgrG have been postulated to form an extracellular translocon. This model is further supported by two recent crystallography studies showing that Hcp, VgrG, and a T4 phage gp25-like protein resembled membrane penetration tails of bacteriophages (35, 45).Little is known about the topology and structure of T6SS machinery subunits and the distinction between genes encoding machinery subunits and genes encoding regulatory proteins. Posttranslational regulation via the phosphorylation of Fha1 by a serine-threonine kinase (PpkA) is required for Hcp secretion from Pseudomonas aeruginosa (42). Genetic evidence for P. aeruginosa suggested that the T6SS may utilize a ClpV-like AAA⁺ ATPase to provide the energy for machinery assembly or substrate translocation (41). A recent study of V. cholerae suggested that ClpV ATPase activity is responsible for remodeling the VipA/VipB tubules which are crucial for type VI substrate secretion (6). An outer membrane lipoprotein, SciN, is an essential T6SS component for mediating Hcp secretion from enteroaggregative Escherichia coli (1). A systematic study of the T6SS machinery in E. tarda revealed that 13 of 16 genes in the evp gene cluster are essential for secretion of T6S substrates (74), which suggests the core components of the T6SS. Interestingly, most of the core components conserved in T6SS are predicted soluble proteins without recognizable signal peptide and transmembrane (TM) domains.The intracellular multiplication F (IcmF) and H (IcmH) proteins are among the few core components with obvious TM domains (8). In Legionella pneumophila Dot/Icm T4SSb, IcmF and IcmH are both membrane localized and partially required for L. pneumophila replication in macrophages (58, 70, 75). IcmF and IcmH are thought to interact with each other in stabilizing the T4SS complex in L. pneumophila (58). In T6SS, IcmF is one of the essential components required for secretion of Hcp from several animal pathogens, including V. cholerae (48), Aeromonas hydrophila (63), E. tarda (74), and P. aeruginosa (41), as well as the plant pathogens A. tumefaciens (72) and Pectobacterium atrosepticum (39). In E. tarda, IcmF (EvpO) interacted with IcmH (EvpN), EvpL, and EvpA in a yeast two-hybrid assay, and its putative nucleotide-binding site (Walker A motif) was not essential for secretion of T6SS substrates (74).In this study, we characterized the topology and interactions of the IcmF and IcmH family proteins ImpL_M and ImpK_L, which are two essential components of the T6SS of A. tumefaciens. We adapted the nomenclature proposed by Cascales (8), using the annotated gene designation followed by the letter indicated by Shalom et al. (59). Our data indicate that ImpL_M and ImpK_L are both integral inner membrane proteins and interact with each other via their N-terminal domains residing in the cytoplasm. We also provide genetic evidence showing that ImpL_M may function as a nucleoside triphosphate (NTP)-binding protein or nucleoside triphosphatase to mediate T6S machinery assembly and/or substrate secretion.     

Cytoskeletal Asymmetrical Dumbbell Structure of a Gliding Mycoplasma,Mycoplasma gallisepticum,Revealed by Negative-Staining Electron Microscopy

Several mycoplasma species feature a membrane protrusion at a cell pole, and unknown mechanisms provide gliding motility in the direction of the pole defined by the protrusion. Mycoplasma gallisepticum, an avian pathogen, is known to form a membrane protrusion composed of bleb and infrableb and to glide. Here, we analyzed the gliding motility of M. gallisepticum cells in detail. They glided in the direction of the bleb at an average speed of 0.4 μm/s and remained attached around the bleb to a glass surface, suggesting that the gliding mechanism is similar to that of a related species, Mycoplasma pneumoniae. Next, to elucidate the cytoskeletal structure of M. gallisepticum, we stripped the envelopes by treatment with Triton X-100 under various conditions and observed the remaining structure by negative-staining transmission electron microscopy. A unique cytoskeletal structure, about 300 nm long and 100 nm wide, was found in the bleb and infrableb. The structure, resembling an asymmetrical dumbbell, is composed of five major parts from the distal end: a cap, a small oval, a rod, a large oval, and a bowl. Sonication likely divided the asymmetrical dumbbell into a core and other structures. The cytoskeletal structures of M. gallisepticum were compared with those of M. pneumoniae in detail, and the possible protein components of these structures were considered.Mycoplasmas are commensal and occasionally pathogenic bacteria that lack a peptidoglycan layer (50). Several species feature a membrane protrusion at a pole; for Mycoplasma mobile, this protrusion is called the head, and for Mycoplasma pneumoniae, it is called the attachment organelle (25, 34-37, 52, 54, 58). These species bind to solid surfaces, such as glass and animal cell surfaces, and exhibit gliding motility in the direction of the protrusion (34-37). This motility is believed to be essential for the mycoplasmas'' pathogenicity (4, 22, 27, 36). Recently, the proteins directly involved in the gliding mechanisms of mycoplasmas were identified and were found to have no similarities to those of known motility systems, including bacterial flagellum, pilus, and slime motility systems (25, 34-37).Mycoplasma gallisepticum is an avian pathogen that causes serious damage to the production of eggs for human consumption (50). The cells are pear-shaped and have a membrane protrusion, consisting of the so-called bleb and infrableb (29), and gliding motility (8, 14, 22). Their putative cytoskeletal structures may maintain this characteristic morphology because M. gallisepticum, like other mycoplasma species, does not have a cell wall (50). In sectioning electron microscopy (EM) studies of M. gallisepticum, an intracellular electron-dense structure in the bleb and infrableb was observed, suggesting the existence of a cytoskeletal structure (7, 24, 29, 37, 58). Recently, the existence of such a structure has been confirmed by scanning EM of the structure remaining after Triton X-100 extraction (13), although the details are still unclear.A human pathogen, M. pneumoniae, has a rod-shaped cytoskeletal structure in the attachment organelle (9, 15, 16, 31, 37, 57). M. gallisepticum is related to M. pneumoniae (63, 64), as represented by 90.3% identity between the 16S rRNA sequences, and it has some open reading frames (ORFs) homologous to the component proteins of the cytoskeletal structures of M. pneumoniae (6, 17, 48). Therefore, the cytoskeletal structures of M. gallisepticum are expected to be similar to those of M. pneumoniae, as scanning EM images also suggest (13).The fastest-gliding species, M. mobile, is more distantly related to M. gallisepticum; it has novel cytoskeletal structures that have been analyzed through negative-staining transmission EM after extraction by Triton X-100 with image averaging (45). This method of transmission EM following Triton X-100 extraction clearly showed a cytoskeletal “jellyfish” structure. In this structure, a solid oval “bell,” about 235 nm wide and 155 nm long, is filled with a 12-nm hexagonal lattice. Connected to this bell structure are dozens of flexible “tentacles” that are covered with particles 20 nm in diameter at intervals of about 30 nm. The particles appear to have 180° rotational symmetry and a dimple at the center. The involvement of this cytoskeletal structure in the gliding mechanism was suggested by its cellular localization and by analyses of mutants lacking proteins essential for gliding.In the present study, we applied this method to M. gallisepticum and analyzed its unique cytoskeletal structure, and we then compared it with that of M. pneumoniae.     

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.     

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.