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).     

New Vector System for Random,Single-Step Integration of Multiple Copies of DNA into the Rhodococcus Genome

We designed a new vector system for creating a random mutant library with multiple integrations of DNA fragments into the Rhodococcus genome in a single step. For this, we cotransformed two vectors into Rhodococcus by electroporation: pTip-istAB-sacB regulates the expression of the transposase (IstA) and its helper protein (IstB) under the influence of a thiostrepton-inducible promoter, and pRTSK-sacB provides the transposable-marker DNA. Both are multicopy vectors that are stable in the host cells; transposition of the transposable-marker DNA occurs only after the induction of IstA/IstB expression. With the addition of thiostrepton, all cultured cells harboring the two vectors, irrespective of the volume, can be mutated by random insertion of the transposable-marker DNA into their genome. Among the generated mutants examined, 30% showed multiple (two to five) insertion copies. The multiple integrated DNA copies were stable in the genome for more than 80 generations of serial growth without the addition of any selective antibiotics. This system can also be used for integrating various copy numbers of stably maintained protein expression cassettes in the host cell genome to modulate the expression level of biologically active recombinant proteins. We successfully applied this system to integrate multiple copies of expression cassettes for proline iminopeptidase and vitamin D₃ hydroxylase into the Rhodococcus genome and verified that the clones containing double or multiple copies of the integrated cassettes produced higher levels and showed higher enzymatic activities of the target protein than clones with only a single copy of integration.The actinobacteria or actinomycetes are a group of Gram-positive bacteria with a high G+C content. Many species of actinobacteria are well known as attractive hosts for the production of biologically active compounds since they can easily utilize cheap complex industrial media and possess excellent secretion capacities. This group includes several antibiotic producers (12, 15) and manufacturers of enzymes (5), amino acids (11), and heterologous proteins (3); hence, they are of high industrial, pharmacological, and commercial interest. Among the genera in this group are Corynebacterium, Mycobacterium, Streptomyces, Nocardia, and Rhodococcus.While a few Rhodococcus species are pathogenic, most are benign and have been found to thrive in a broad range of environments, including soil, water, and eukaryotic cells. Rhodococcus is an experimentally advantageous system due to its relatively fast growth rate and simple developmental cycle. Rhodococcus erythropolis can grow at temperatures ranging from 4 to 35°C (41), which enables the investigation of protein production over a wide range of temperatures (24). Strains of Rhodococcus have important applications due to their ability to bioconvert cheap starting material into more valuable compounds (23) and to metabolize harmful environmental pollutants such as toluene, naphthalene, herbicides, and polychlorinated biphenyls (PCBs) (6, 17). This genetic and catabolic diversity of Rhodococcus is the result of not only its large bacterial chromosome but also the presence of large linear plasmids (37). To date, 43 species of Rhodococcus (7; reference periodically updated at http://www.bacterio.net.) have been recognized (http://www.bacterio.cict.fr/qr/rhodococcus.html). However, Rhodococcus is not yet fully characterized.Various genetic tools have been established for the genetic manipulation of Rhodococcus. These include the development of efficient transformation techniques using electroporation (33); construction of expression vectors for protein production (24, 25); and development of shuttle vectors using cryptic, antibiotic-resistant, and temperature-sensitive plasmids (16, 18, 19, 22) derived from Rhodococcus strains as well as the generation of random mutagenesis using transposons.Several transposon mutagenesis systems have been reported for Rhodococcus species (1, 8, 20, 21). These systems can generate a single copy of insertion into the host cell genome. To date, no efficient tool is available for the creation of random multiple gene disruptions in a single step. Previous researches on the creation of multiple integrations in a genome have been based on site-directed mutagenesis or gene disruption in sequential steps, which requires different antibiotic markers for mutant selection (9, 28, 31, 36, 39, 40).We recently established the transposon-based vector system pTNR that can efficiently generate a random mutagenesis library by transposition in various Rhodococcus species (30). Inside the Rhodococcus cell, pTNR is unstable due to the lack of a replication origin for Rhodococcus. The expression of the transposase (IstA) and its helper protein (IstB) in pTNR is regulated under the influence of the constitutive promoter, P_nit (25). Once pTNR is electroporated into Rhodococcus cells, transposition occurs, whereby IstA and IstB are simultaneously expressed and initiate the integration of a single copy of the transposable-marker DNA into the host cell genome while the rest of the plasmid itself is lost. The transposable-marker DNA of pTNR locates between the two inverted repeats, IR1 and IR2, and encodes a replication origin for Escherichia coli and a kanamycin resistance gene, enabling easy identification of the insertion site via plasmid rescue from the genome (30).pTNR was further modified to be used for protein expression through insertion of the protein expression cassette into the host cell genome (29). Currently, four variants of pTNR vectors are available, each with a different antibiotic resistance marker gene. Two or more variants of pTNR can be used for creating double or multiple integrations in sequential steps or even in one step if the variants are cotransposed in combinations. Nonetheless, the incidence of achieving double or multiple integrations from the cotransposed pTNR variants is very low; hence, the statistical chance of inactivating multiple genes within a definite metabolic pathway or bioprocess by such a method is extremely low. To knock out these functionally related genes, an effective method to produce huge numbers of mutations with random insertions at multiple loci is required. To that end, the present study aimed to develop an efficient genome engineering system for random integration of multiple DNA copies into the Rhodococcus genome in a single step.     

Effects of Ammonium and Nitrite on Growth and Competitive Fitness of Cultivated Methanotrophic Bacteria

The effects of nitrite and ammonium on cultivated methanotrophic bacteria were investigated. Methylomicrobium album ATCC 33003 outcompeted Methylocystis sp. strain ATCC 49242 in cultures with high nitrite levels, whereas cultures with high ammonium levels allowed Methylocystis sp. to compete more easily. M. album pure cultures and cocultures consumed nitrite and produced nitrous oxide, suggesting a connection between denitrification and nitrite tolerance.The application of ammonium-based fertilizers has been shown to immediately reduce the uptake of methane in a number of diverse ecological systems (3, 5, 7, 8, 11-13, 16, 27, 28), due likely to competitive inhibition of methane monooxygenase enzymes by ammonia and production of nitrite (1). Longer-term inhibition of methane uptake by ammonium has been attributed to changes in methanotrophic community composition, often favoring activity and/or growth of type I Gammaproteobacteria methanotrophs (i.e., Gammaproteobacteria methane-oxidizing bacteria [gamma-MOB]) over type II Alphaproteobacteria methanotrophs (alpha-MOB) (19-23, 25, 26, 30). It has been argued previously that gamma-MOB likely thrive in the presence of high N loads because they rapidly assimilate N and synthesize ribosomes whereas alpha-MOB thrive best under conditions of N limitation and low oxygen levels (10, 21, 23).Findings from studies with rice paddies indicate that N fertilization stimulates methane oxidation through ammonium acting as a nutrient, not as an inhibitor (2). Therefore, the actual effect of ammonium on growth and activity of methanotrophs depends largely on how much ammonia-N is used for assimilation versus cometabolism. Many methanotrophs can also oxidize ammonia into nitrite via hydroxylamine (24, 29). Nitrite was shown previously to inhibit methane consumption by cultivated methanotrophs and by organisms in soils through an uncharacterized mechanism (9, 17, 24), although nitrite inhibits purified formate dehydrogenase from Methylosinus trichosporium OB3b (15). Together, the data from these studies show that ammonium and nitrite have significant effects on methanotroph activity and community composition and reveal the complexity of ammonia as both a nutrient and a competitive inhibitor. The present study demonstrates the differential influences of high ammonium or nitrite loads on the competitive fitness of a gamma-MOB versus an alpha-MOB strain.     

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.     

Isolation and Characterization of “Dehalococcoides” sp. Strain MB,Which Dechlorinates Tetrachloroethene to trans-1,2-Dichloroethene

In an attempt to understand the microorganisms involved in the generation of trans-1,2-dichloroethene (trans-DCE), pure-culture “Dehalococcoides” sp. strain MB was isolated from environmental sediments. In contrast to currently known tetrachloroethene (PCE)- or trichloroethene (TCE)-dechlorinating pure cultures, which generate cis-DCE as the predominant product, Dehalococcoides sp. strain MB reductively dechlorinates PCE to trans-DCE and cis-DCE at a ratio of 7.3 (±0.4):1. It utilizes H₂ as the sole electron donor and PCE or TCE as the electron acceptor during anaerobic respiration. Strain MB is a disc-shaped, nonmotile bacterium. Under an atomic force microscope, the cells appear singly or in pairs and are 1.0 μm in diameter and ∼150 nm in depth. The purity was confirmed by culture-based approaches and 16S rRNA gene-based analysis and was corroborated further by putative reductive dehalogenase (RDase) gene-based, quantitative real-time PCR. Although strain MB shares 100% 16S rRNA gene sequence identity with Dehalococcoides ethenogenes strain 195, these two strains possess different dechlorinating pathways. Microarray analysis revealed that 10 putative RDase genes present in strain 195 were also detected in strain MB. Successful cultivation of strain MB indicates that the biotic process could contribute significantly to the generation of trans-DCE in chloroethene-contaminated sites. It also enhances our understanding of the evolution of this unusual microbial group, Dehalococcoides species.Dehalorespiring bacteria play an important role in the transformation and detoxification of a wide range of halogenated compounds, e.g., chlorophenols, chloroethenes, chlorobenzenes, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs) (2, 4, 9, 14, 16, 17, 32, 35, 38). Among these compounds, the organic solvents tetrachloroethene (PCE) and trichloroethene (TCE) are suspected carcinogens that are found in soil and groundwater due to their extensive usage and improper disposal (6). The widespread PCE and TCE in the subsurface environment have driven intensive studies of anaerobic microbes capable of reductive dechlorination of chloroethenes (40). Over the last decade, at least 18 isolates, which belong to the genera Desulfitobacterium, Sulfurospirillum, Desulfomonile, Desulfuromonas, Geobacter, “Dehalococcoides,” and Dehalobacter, show reductive dechlorination of chlorinated ethenes (16, 40). In particular, most of these microbes produce cis-1,2-dichloroethene (cis-DCE) as the end product in the chloroethene-contaminated sites, whereas complete detoxification of PCE or TCE to ethene has been restricted only to members of the genus Dehalococcoides. Thus, the Dehalococcoides species have received considerable attention from the bioremediation community in the past decade.Several strains of Dehalococcoides species (e.g., 195, CBDB1, BAV1, and VS) have been sequenced for their whole genomes (24, 39). Their dechlorinating capabilities have also been well addressed through identification and quantification of the known chloroethene reductive dehalogenase (RDase) genes or expression of specific RDase genes (18, 21, 25, 41). In chloroethene-contaminated sites, the natural activities of single or multiple Dehalococcoides strains can lead either to more-toxic, mobile intermediates (e.g., cis- or trans-DCEs and vinyl chloride [VC]) via partial dechlorination of PCE/TCE or to harmless ethene by complete detoxification (10, 13, 15, 41). Many mixed cultures and pure isolates have been reported to produce cis-DCE or VC during PCE/TCE dechlorination processes (15, 40, 43). However, trans-DCE has been detected in more than one-third of the U.S. Environmental Protection Agency (EPA) superfund sites (3a). The source of trans-DCE production was thought to be an abiotic process; however, recently both trans-DCE generation and cis-DCE generation were reported to occur via microbial dechlorination.To date, microbes from either Dehalococcoides- or DF-1-containing mixed cultures have been reported to produce more trans- than cis-DCE, with a ratio of 1.2:1 to 3.5:1 in laboratory-scale studies (8, 10, 22, 31). For example, in a recent report by Kittelmann and Friedrich (22), trans-/cis-DCE at a ratio of 3.5:1 was generated in tidal flat sediment-containing microcosms with microbes closely related to Dehalococcoides sp. or DF-1-like microbes. Additionally, Griffin et al. identified Dehalococcoides species of the Pinellas subgroup in several enrichment cultures, which dechlorinated TCE (∼0.25 mM) to trans-DCE and cis-DCE at a ratio of ∼3:1 (10). There is no information available on the Dehalococcoides isolates that generate trans-DCE as the main end product. This also means a lack of information on the genomic contents of trans-DCE-producing bacteria. Therefore, finding microorganisms that produce trans-DCE in pure culture will be useful for the comprehensive characterization of this group of bacteria.The aim of this study was to isolate a PCE-to-trans-DCE-dechlorinating culture to facilitate the elucidation of trans-DCE formation during reductive dechlorination processes. Microarray analysis was conducted to compare the whole-genome contents of the new isolate and the well-characterized Dehalococcoides ethenogenes strain 195 (30). In addition, a coculture which consisted of the new isolate and TCE-to-cis-DCE-to-VC-dechlorinating Dehalococcoides sp. strain ANAS1 was explored to study the interaction, distribution, and function of the dechlorinators in the dechlorinating process.     

Lateral Transfer of Genes for Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) Degradation

Recent studies demonstrated that degradation of the military explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by species of Rhodococcus, Gordonia, and Williamsia is mediated by a novel cytochrome P450 with a fused flavodoxin reductase domain (XplA) in conjunction with a flavodoxin reductase (XplB). Pulse field gel analysis was used to localize xplA to extrachromosomal elements in a Rhodococcus sp. and distantly related Microbacterium sp. strain MA1. Comparison of Rhodococcus rhodochrous 11Y and Microbacterium plasmid sequences in the vicinity of xplB and xplA showed near identity (6,710 of 6,721 bp). Sequencing of the associated 52.2-kb region of the Microbacterium plasmid pMA1 revealed flanking insertion sequence elements and additional genes implicated in RDX uptake and degradation.Past practices of production, application, and disposal of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) have resulted in widespread contamination. Environmental contamination is aggravated by its high mobility, contributing to more-widespread contamination of groundwater than by other commonly used explosives (27). Ingestion or inhalation of RDX is associated with neurological disorders and organ failure (38), and exposed wildlife show behavioral changes and suffer liver and reproductive damage (38). The U.S. Environmental Protection Agency (EPA) has classified RDX as a possible human carcinogen (37). These adverse effects have provided motivation to better understand the microbiology and biochemistry of RDX degradation.As yet there is relatively limited information concerning natural rates of microbial RDX degradation or degradation mechanisms; such information is needed to predict or control rates of degradation in the environment. Of the three general pathways for RDX degradation or transformation based on metabolite analysis outlined in the review by Crocker and associates (6), aerobic degradation initiated by XplA is among the better-characterized systems. This enzyme, a novel cytochrome P450 with a fused flavodoxin reductive domain (18, 33), was first identified by Seth-Smith et al. in Rhodococcus rhodochrous 11Y as being encoded by xplA (33). This gene has been identified in 24 bacterial isolates of the Corynebacterineae capable of utilizing RDX as a sole nitrogen source (4, 25, 32-34). While mammalian nitric oxide synthase family enzymes are known to be P450-like enzymes with fused flavodoxin domains, there are very few identified examples of this type of protein fusion among characterized microbial species (2, 15, 18, 24). Subsequent studies by Jackson and associates (18) demonstrated that XplA, in association with an electron-transferring flavodoxin reductase (XplB), functions to efficiently denitrate RDX aerobically to the aliphatic nitramine 4-nitro-2,4-diazabutanal (NDAB) (18). NDAB has been shown to serve as a viable nitrogen source for Methylobacterium sp. strain JS178 (13) and to be degraded by Phanerochaete chrysosporium (11). Thus, complete mineralization often appears to be mediated by multiple microbial populations.The capacity for microbial degradation of recalcitrant organics, many of which are apparently new to the biosphere as a result of chemical manufacture, is often determined by plasmids and associated mobile genetic elements (36, 39). Plasmids both serve as a reservoir of genetic information and promote metabolic innovation, since their replication is independent of the chromosome and they do not generally encode essential functions. Although it was earlier suggested that genes in Rhodococcus sp. strain DN22 associated with initial steps of RDX degradation are carried by plasmids (5), no direct evidence for an extrachromosomal location was provided. We now show that nearly identical genes for XplA and XplB are carried on plasmids in two phylogenetically and geographically distinct bacterial isolates: Microbacterium sp. strain MA1, isolated from North America (Milan, TN), and Rhodococcus rhodochrous 11Y, isolated from England (33). Thus, these genes are more broadly distributed within the Actinomycetales than previously recognized, and the near identity of gene sequence (6,710 of 6,721 bp) in these divergent genera is indicative of recent plasmid-mediated transfer. Analysis of approximately 52 kbp of sequence near xplA and xplB in strain MA1 revealed closely linked genes for transport and degradation that are flanked by transposable elements, suggesting that plasmid-carried xplA and xplB are part of a larger class I transposable element encoding both transport and degradation of RDX.     

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.     

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.     

Protistan Predation Affects Trichloroethene Biodegradation in a Bedrock Aquifer

Despite extensive research on the bottom-up force of resource availability (e.g., electron donors and acceptors), slow biodegradation rates and stalling at cis-dichloroethene (cDCE) and vinyl chloride continue to be observed in aquifers contaminated with trichloroethene (TCE). The objective of this research was to gauge the impact of the top-down force of protistan predation on TCE biodegradation in laboratory microcosms. When indigenous bacteria from an electron donor-limited TCE-contaminated bedrock aquifer were present, the indigenous protists inhibited reductive dechlorination altogether. The presence of protists during organic carbon-amended conditions caused the bacteria to elongate (length:width, ≥10:1), but reductive dechlorination was still inhibited. When a commercially available dechlorinating bacterial culture and an organic carbon amendment were added in he presence of protists, the elongated bacteria predominated and reductive dechlorination stalled at cDCE. When protists were removed under organic carbon-amended conditions, reductive dechlorination stalled at cDCE, whereas in the presence organic carbon and bacterial amendments, the total chlorinated ethene concentration decreased, indicating TCE was converted to ethene and/or CO₂. The data suggested that indigenous protists grazed dechlorinators to extremely low levels, inhibiting dechlorination altogether. Hence, in situ bioremediation/bioaugmentation may not be successful in mineralizing TCE unless the top-down force of protistan predation is inhibited.The bacterially mediated sequential dechlorination of trichloroethene (TCE) to cis-dichloroethene (cDCE), vinyl chloride (VC), ethene, and CO₂ by dehalorespiration is often proposed as the most cost-effective in situ treatment to remediate chlorinated solvent-contaminated aquifers (35, 42). TCE mineralization to CO₂ requires specific electron donors (i.e., acetate and H₂) typically produced from readily fermentable organic carbon, the presence of specific bacterial species, and sulfate-reducing or methanogenic conditions (1, 4, 8, 15, 22, 25, 33, 35, 46). When the rate of mineralization is slow or stalled at one of the progeny (cDCE and VC), the problem is usually attributed to the bottom-up force of resource availability (e.g., the absence of a necessary condition such as suitable electron donors or bacterial species) (1, 4, 10, 22, 26, 43, 46). For example, whereas many bacterial species are capable of degrading TCE to cDCE and VC by dehalorespiration (33), only Dehalococcoides ethenogenes is known to convert VC to ethene (25). Hence, if an indigenous population of D. ethenogenes is not present in situ, the system will likely stall at cDCE or VC even if sufficient electron donor is added. Stalling is problematic because VC is more toxic than TCE (18). In this case, bioaugmentation with D. ethenogenes may trigger complete mineralization.An established link exists for the top-down force of predator-prey relationships between protists and bacteria in a range of surface water systems (13, 19-21, 29). Our previous work with groundwater protists in a wastewater contaminated sandy aquifer demonstrated that size selective predation by protists affects biodegradation of the organic carbon. Our subsequent work in a TCE-contaminated bedrock aquifer at the Bedrock Bioremediation Center (BBC) research site (Portsmouth, NH) suggested that bottom-up resource availability could not totally explain stalls at cDCE. This led us to hypothesize that the top-down force of selective predation by protists on dehalorespiring bacteria inhibited the required compositional shift in the bacterial community to one dominated by D. ethenogenes, thus preventing the conversion of cDCE and VC to ethene and CO₂ (2, 15). This, coupled with bacterial studies by Travis and Rosenberg (41), Lewis (32) and Snyder et al. (40), led us to postulate that protistan predation could have a negative impact on TCE biodegradation. Continuously stirred reactors were used to examine how the presence of protists influenced the rate of bacterially mediated reductive dechlorination. Experiments were conducted using ambient (≤0.8 mM as C) and amended (10 mM as C) organic carbon concentrations with protists present and absent. TCE biodegradation was also assessed when the indigenous community was amended with a commercially available bacterial culture containing D. ethenogenes (34).(A portion of this research was originally submitted to the University of New Hampshire, Durham, by J. Cunningham as an M.S. thesis [12].)     

Actions of Mycobacterium sp. Strain AP1 on the Saturated- and Aromatic-Hydrocarbon Fractions of Fuel Oil in a Marine Medium

The pyrene-degrading Mycobacterium sp. strain AP1 grew in nutrient-supplemented artificial seawater with a heavy fuel oil as the sole carbon source, causing the complete removal of all linear (C₁₂ to C₄₀) and branched alkanes from the aliphatic fraction, as well as an extensive degradation of the three- and four-ring polycyclic aromatic hydrocarbons (PAHs) phenanthrene (95%), anthracene (80%), fluoranthene (80%), pyrene (75%), and benzo(a)anthracene (30%). Alkylated PAHs, which are more abundant in crude oils than the nonsubstituted compounds, were selectively attacked at extents that varied from more than 90% for dimethylnaphthalenes, methylphenanthrenes, methylfluorenes, and methyldibenzothiophenes to about 30% for monomethylated fluoranthenes/pyrenes and trimethylated phenanthrenes and dibenzothiophenes. Identification of key metabolites indicated the utilization of phenanthrene, pyrene, and fluoranthene by known assimilatory metabolic routes, while other components were cooxidized. Detection of mono- and dimethylated phthalic acids demonstrated ring cleavage and further oxidation of alkyl PAHs. The extensive degradation of the alkanes, the two-, three-, and four-ring PAHs, and their 1-, 2-, and 3-methyl derivatives from a complex mixture of hydrocarbons by Mycobacterium sp. strain AP1 illustrates the great substrate versatility of alkane- and PAH-degrading mycobacteria.Accidental oil spills cause extensive ecological damage to marine shorelines and also have an enormous impact on related economic activities due to the potential risk to public health. One of the most recent examples is the heavy fuel oil spill from the tanker Prestige in 2002, which affected 1,900 km of coast in northwestern Spain. While the light fractions of the oil evaporate in the early stages of a spill, microbial degradation plays a major role in the removal of the heavier fractions. Stimulation of natural biodegradation processes by nutrient and fertilizer addition has proven to enhance oil degradation in a variety of coastal environments (3, 42, 44).Oil is a complex mixture of hundreds of components that can be separated into saturates, aromatics, resins, and asphaltenes. The saturated hydrocarbons are usually the most abundant, while polycyclic aromatic hydrocarbons (PAHs) cause the greatest concern because of their toxic and genotoxic potentials.Most of the available knowledge on the microbial processes involved in PAH biodegradation has been obtained from studies involving bacterial isolates acting on single substrates that serve as the sole source of carbon and energy for growth (7, 20, 22). The pathways for the complete degradation of hydrocarbons containing two and three aromatic rings by gram-negative bacteria are well characterized for such conditions (7, 22). Conversely, degradation of hydrocarbons containing four or more fused aromatic rings, such as pyrene, has been reported only for soil actinomycetes (20, 25, 29, 30, 36, 45), which use multibranched pathways involving both classical dioxygenation and meta-cleavage reactions and novel ortho-cleavage mechanisms uncommon in gram-negative organisms (23). Due to the relaxed specificity of some degradative enzymes, mainly dioxygenases (15, 37), PAH-degrading strains have a wide range of substrates, being able to act simultaneously on a number of structural analogs and to oxidize them to different extents (18, 37). However, the individual processes involved in the degradation of naturally occurring complex mixtures of PAHs (crude oils and coal derivatives) have rarely been addressed (18, 31).Early studies on biodegradation of crude oil were carried out with bacterial strains able to use this mixture for growth. Since PAHs and other components are contained within a predominantly aliphatic matrix in crude oil, most of these studies reported actions of alkane degraders on individual oil components (2, 34, 38, 41, 50). In addition to alkanes, these alkane degraders selectively depleted some alkylated PAHs (2, 41), a process that has been attributed to partial oxidation due to a monooxygenase attack on the methyl groups to produce the corresponding carboxylic acids (35). Recent studies reported the isolation of a number of two- and three-ring-PAH-degrading bacterial strains from coastal sediments affected by crude oil spills. These strains include members of genera commonly isolated from PAH-contaminated soils, such as Pseudomonas (39, 43) and Sphingomonas (49), as well as less common genera, such as Marinobacter (13), Moraxella (43), Vibrio (51), and Cycloclasticus (12). The last genus seems to play a major role in the fate of low-molecular-weight PAHs in the marine environment, as members of this genus have been isolated from several crude oil-contaminated locations (6, 14, 21). When incubated with crude oil, Cycloclasticus strains degraded most of the two- and three-ring PAHs and some of their alkyl derivatives (C_0-4 naphthalene, C_0-2 dibenzothiophene, C_0-2 phenanthrene, and C_0-2 fluorene [numerals indicate the number of methyl groups]). However, neither alkanes, trimethyl derivatives of three-ring PAHs, or higher-molecular-weight PAHs were significantly depleted (21). On the other hand, no attempts were made to identify metabolic intermediates indicative of specific degradation or cometabolic pathways.Alkyl-PAH degradation is isomer specific, a feature that has been used in geochemistry to define source recognition and oil weathering ratios (47). For example, given the resistance of 9-methyl phenanthrene to microbial oxidation in relation to the other isomers, the ratio of 3-methylphenanthrene plus 2-methylphenanthrene to 9-methylphenanthrene plus 1-methylphenanthrene has been utilized as a diagnostic ratio (47). These ratios have been defined on the basis of analysis of environmental samples (47) and results of crude oil biodegradation assays with mixed cultures (10, 48) or single strains (2, 41), mainly alkane-degrading pseudomonads. The actions of high-molecular-weight-PAH-degrading mycobacteria on the alkylated families of PAHs present in crude oil and derivatives have not been addressed.Mycobacterium strains isolated by their ability to grow on pyrene have often been shown to also utilize phenanthrene, fluoranthene, and high-molecular-weight alkanes as single carbon sources (8, 45). In a recent study, we showed that when Mycobacterium strain AP1, isolated from an oil-polluted marine beach, was incubated with a mixture of PAHs from creosote, this strain caused a significant depletion of the three-aromatic-ring PAHs but had a limited action on the higher-molecular-weight PAHs fluoranthene and pyrene (31). Given the wide substrate versatility of pyrene-degrading mycobacteria, especially for alkane degradation, their presence in marine environments (16), and their distinctive reactions during PAH degradation (22, 25, 30), in this study we used strain AP1 to investigate the catabolic potential of mycobacteria in the removal of the most abundant hydrocarbon families and their derivatives from crude oil in a marine medium under laboratory conditions. The identification of key metabolites indicative of previously proposed reactions gave insight into the metabolic and cometabolic processes involved. As a model mixture, we used the heavy fuel oil spilled from the Prestige, a Russian M100 fuel oil especially rich in aromatic hydrocarbons (52%) (27).     

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.     

Population Structure of the Lyme Borreliosis Spirochete Borrelia burgdorferi in the Western Black-Legged Tick (Ixodes pacificus) in Northern California

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.In the United States, Lyme borreliosis (LB) is the most commonly reported vector-borne illness and is caused by infection with the spirochete Borrelia burgdorferi (3, 9, 52). The signs and symptoms of LB can include a rash, erythema migrans, fever, fatigue, arthritis, carditis, and neurological manifestations (50, 51). The black-legged tick, Ixodes scapularis, and the western black-legged tick, Ixodes pacificus, are the primary vectors of B. burgdorferi to humans in the United States, with the former in the northeastern and north-central parts of the country and the latter in the Far West (9, 10). These ticks perpetuate enzootic transmission cycles together with a vertebrate reservoir host such as the white-footed mouse, Peromyscus leucopus, in the Northeast and Midwest (24, 35), or the western gray squirrel, Sciurus griseus, in California (31, 46).B. burgdorferi is a spirochete species with a largely clonal population structure (14, 16) comprising several different strains or lineages (8). The polymorphic ospC gene of B. burgdorferi encodes a surface lipoprotein that increases expression within the tick during blood feeding (47) and is required for initial infection of mammalian hosts (25, 55). To date, approximately 20 North American ospC genotypes have been described (40, 45, 49, 56). At least four, and possibly up to nine, of these genotypes are associated with B. burgdorferi invasiveness in humans (1, 15, 17, 49, 57). Restriction fragment length polymorphism (RFLP) and, subsequently, sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) are used as molecular typing tools to investigate genotypic variation in B. burgdorferi (2, 36, 38, 44, 44, 57). The locus maintains a high level of variation between related species, and this variation reflects the heterogeneity found at the genomic level of the organism (37). The IGS and ospC loci appear to be linked (2, 8, 26, 45, 57), but the studies to date have not been representative of the full range of diversity of B. burgdorferi in North America.Previous studies in the northeastern and midwestern United States have utilized IGS and ospC genotyping to elucidate B. burgdorferi evolution, host strain specificity, vector-reservoir associations, and disease risk to humans. In California, only six ospC and five IGS genotypes have been described heretofore in samples from LB patients or I. pacificus ticks (40, 49, 56) compared to approximately 20 ospC and IGS genotypes identified in ticks, vertebrate hosts, or humans from the Northeast and Midwest (8, 40, 45, 49, 56). Here, we employ sequence analysis of both the ospC gene and IGS region to describe the population structure of B. burgdorferi in more than 200 infected I. pacificus nymphs from Mendocino County, CA, where the incidence of LB is among the highest in the state (11). Further, we compare the Mendocino County spirochete population to populations found in the Northeast.