Responses of Ammonia-Oxidizing Bacterial and Archaeal Populations to Organic Nitrogen Amendments in Low-Nutrient Groundwater

To evaluate the potential for organic nitrogen addition to stimulate the in situ growth of ammonia oxidizers during a field scale bioremediation trial, samples collected from the Eastern Snake River Plain Aquifer in Idaho before, during, and after the addition of molasses and urea were subjected to PCR analysis of ammonia monooxygenase subunit A (amoA) genes. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) were present in all of the samples tested, with AOA amoA genes outnumbering AOB amoA genes in all of the samples. Following urea addition, nitrate levels rose and bacterial amoA copy numbers increased dramatically, suggesting that urea hydrolysis stimulated nitrification. Bacterial amoA diversity was limited to two Nitrosomonas phylotypes, whereas archaeal amoA analyses revealed 20 distinct operational taxonomic units, including several that were markedly different from all previously reported sequences. Results from this study demonstrate the likelihood of stimulating ammonia-oxidizing communities during field scale manipulation of groundwater conditions to promote urea hydrolysis.Subsurface calcite precipitation driven by microbial urea hydrolysis has been proposed as a means of remediating trace metal or radionuclide contaminants (e.g., strontium-90) that can be coprecipitated and retained in the solid phase (11, 12, 42). Urea hydrolysis generates carbonate alkalinity and raises pH, both of which promote calcite precipitation. However, another product of urea hydrolysis is ammonium, as shown in the following equation: In low-nutrient groundwater, the ammonium resulting from urea hydrolysis can have a number of fates, including uptake by nitrogen-limited microorganisms or transformation to nitrite by ammonia-oxidizing microorganisms. Microbial oxidation of ammonia is a net acid-yielding process. The resultant acidity from this reaction could inhibit calcite precipitation or promote destabilization of preexisting calcite, potentially liberating contaminants from the solid phase. In addition, the further transformation of nitrite by nitrite-oxidizing bacteria leads to the formation of nitrate, a regulated contaminant of drinking water.The first step of bacterial ammonia oxidation, the conversion of ammonia to hydroxylamine, is catalyzed by the membrane-bound enzyme ammonia monooxygenase. The gene coding for the catalytic α subunit of this enzyme, amoA, has proven to be an effective molecular marker for ammonia-oxidizing bacteria (AOB) (20, 34). All of the currently known chemoautotrophic AOB are associated with the Nitrosomonas and Nitrosospira genera within the Betaproteobacteria or the genus Nitrosococcus within the Gammaproteobacteria (15, 32). Although ammonia oxidation was long believed to be carried out exclusively by members of the domain Bacteria, considerable evidence now suggests that recently discovered ammonia-oxidizing archaea (AOA) (18) are key players in this critical step of the microbial nitrogen cycle (8).The archaeal amoA gene has been found in a wide range of environments (9; reviewed in references 8 and 31), and its expression has been documented in enrichment cultures (35) and soil microcosms (40), as well as in marine and terrestrial environments (21, 23). Reported quantitative PCR (qPCR) analyses of amoA in marine and terrestrial environments suggest that AOA typically outnumber AOB by orders of magnitude (23, 26, 44), and AOA abundance has also recently been shown to be highly correlated with water column ¹⁵NH₄⁺ oxidation rates (1). However, some recent studies have reported that AOB are more abundant under certain conditions (6, 27, 35, 43, 45).In an effort to better understand the fate of ammonium generated from urea hydrolysis, we monitored the abundance and diversity of bacterial and archaeal amoA genes during a field experiment designed to test stimulation of urea hydrolysis in groundwater. Dilute molasses and urea were sequentially introduced into a well in the Eastern Snake River Plain Aquifer (ESRPA) in Idaho (13). Previous laboratory experiments indicated that molasses, an inexpensive and commonly used bioremediation amendment (14), was effective in increasing overall microbial populations, as well as total ureolytic activity (13, 39). The ESRPA is a deep basalt aquifer and is considered oligotrophic (4, 22, 29); however, previous work has demonstrated the presence of ureolytic microbes in this environment (11, 13). Erwin et al. also reported evidence of AOB during the analysis of methane monooxygenase clone libraries from ESRPA samples (7), but in general, the structure and function of ammonia-oxidizing microbial communities (and especially AOA) in deep aquifers like the ESRPA have been relatively unexplored.     

Cultivation of Autotrophic Ammonia-Oxidizing Archaea from Marine Sediments in Coculture with Sulfur-Oxidizing Bacteria

The role of ammonia-oxidizing archaea (AOA) in nitrogen cycling in marine sediments remains poorly characterized. In this study, we enriched and characterized AOA from marine sediments. Group I.1a crenarchaea closely related to those identified in marine sediments and “Candidatus Nitrosopumilus maritimus” (99.1 and 94.9% 16S rRNA and amoA gene sequence identities to the latter, respectively) were substantially enriched by coculture with sulfur-oxidizing bacteria (SOB). The selective enrichment of AOA over ammonia-oxidizing bacteria (AOB) is likely due to the reduced oxygen levels caused by the rapid initial growth of SOB. After biweekly transfers for ca. 20 months, archaeal cells became the dominant prokaryotes (>80%), based on quantitative PCR and fluorescence in situ hybridization analysis. The increase of archaeal 16S rRNA gene copy numbers was coincident with the amount of ammonia oxidized, and expression of the archaeal amoA gene was observed during ammonia oxidation. Bacterial amoA genes were not detected in the enrichment culture. The affinities of these AOA to oxygen and ammonia were substantially higher than those of AOB. [¹³C]bicarbonate incorporation and the presence and activation of genes of the 3-hydroxypropionate/4-hydroxybutyrate cycle indicated autotrophy during ammonia oxidation. In the enrichment culture, ammonium was oxidized to nitrite by the AOA and subsequently to nitrate by Nitrospina-like bacteria. Our experiments suggest that AOA may be important nitrifiers in low-oxygen environments, such as oxygen-minimum zones and marine sediments.Archaea have long been known as extremophiles, since most cultivated archaeal strains were cultivated from extreme environments, such as acidic, hot, and high-salt environments. The view of archaea as extremophiles (i.e., acidophiles, thermophiles, and halophiles) has radically changed by the application of molecular technologies, including PCR in environmental microbiology. Using Archaea-specific PCR primers, novel archaeal 16S rRNA gene sequences were discovered in seawater (23, 27). Following these discoveries, an ever-increasing and unexpectedly high variety of archaeal 16S rRNA gene sequences has been reported from diverse “nonextreme” environments (67). This indicates that archaea are, like bacteria, ubiquitous in the biosphere rather than exclusively inhabiting specific extreme niches. Archaea are abundant in water columns of some oceanic provinces (33, 36) and deep-subsea floor sediments (11, 12, 48). Despite the increasing number of reports of the diversity and abundance of these nonextreme archaea by molecular ecological studies, their physiology and ecological roles have remained enigmatic.Oxidation of ammonia, a trait long thought to be exclusive to the domain Bacteria (13), was recently suggested to be a trait of archaea of the crenarchaeal groups I.1a and I.1b, based on a metagenome analysis (79) and supported by the discovery of archaeal amoA-like genes in environmental shotgun sequencing studies of Sargasso Sea water (80) and genomic analysis of “Candidatus Cenarchaeum symbiosum,” a symbiont of a marine sponge (30). Molecular ecological studies indicated that these ammonia-oxidizing archaea (AOA) are often predominant over ammonia-oxidizing bacteria (AOB) in ocean waters (9, 53, 87), soils (17, 47), and marine sediments (61). Critical evidence for autotrophic archaeal ammonia oxidation was obtained by the characterization of the first cultivated mesophilic crenarchaeon (group I.1a), “Candidatus Nitrosopumilus maritimus SCM1,” from an aquarium (38), and a related archaeon from North Sea water (87) and subsequently by enrichment of thermophilic AOA (22, 31). Whole-genome-based phylogenetic studies recently indicated that the nonthermophilic crenarchaea, including the AOA, likely form a phylum separate from the Crenarchaeota and Euryarchaeota phyla (15, 16, 72). This proposed new phylum was called Thaumarchaeota (15).Microorganisms in marine sediments contribute significantly to global biogeochemical cycles because of their abundance (85). Nitrification is essential to the nitrogen cycle in marine sediments and may be metabolically coupled with denitrification and anaerobic ammonium oxidation, resulting in the removal of nitrogen as molecular nitrogen and the generation of greenhouse gases, such as nitrous oxide (19, 75). Compared with studies on archaeal nitrification in the marine water column, only limited information on archaeal nitrification in marine sediments is available so far. Archaeal amoA genes have been retrieved from marine and coastal sediments (8, 26, 61), and the potentially important role of AOA in nitrification has been suggested based on the abundance of archaeal amoA genes relative to that of bacterial amoA genes in surface marine sediments from Donghae (South Korea) (61). Cultivation of AOA, although difficult (38), remains essential to estimating the metabolic potential of archaea in environments such as soils (47) and marine sediments (61). Here, we report the successful enrichment of AOA of crenarchaeal group I.1a from marine sediments by employing a coculture with sulfur-oxidizing bacteria (SOB) which was maintained for ca. 20 months with biweekly transfers. In this way, we were able to characterize AOA from marine sediments, providing a clue for the role of AOA in the nitrogen cycle of marine sediments.     

Diversity,Abundance, and Spatial Distribution of Sediment Ammonia-Oxidizing Betaproteobacteria in Response to Environmental Gradients and Coastal Eutrophication in Jiaozhou Bay,China

Ongoing anthropogenic eutrophication of Jiaozhou Bay offers an opportunity to study the influence of human activity on bacterial communities that drive biogeochemical cycling. Nitrification in coastal waters appears to be a sensitive indicator of environmental change, suggesting that function and structure of the microbial nitrifying community may be associated closely with environmental conditions. In the current study, the amoA gene was used to unravel the relationship between sediment aerobic obligate ammonia-oxidizing Betaproteobacteria (Beta-AOB) and their environment in Jiaozhou Bay. Protein sequences deduced from amoA gene sequences grouped within four distinct clusters in the Nitrosomonas lineage, including a putative new cluster. In addition, AmoA sequences belonging to three newly defined clusters in the Nitrosospira lineage were also identified. Multivariate statistical analyses indicated that the studied Beta-AOB community structures correlated with environmental parameters, of which nitrite-N and sediment sand content had significant impact on the composition, structure, and distribution of the Beta-AOB community. Both amoA clone library and quantitative PCR (qPCR) analyses indicated that continental input from the nearby wastewater treatment plants and polluted rivers may have significant impact on the composition and abundance of the sediment Beta-AOB assemblages in Jiaozhou Bay. Our work is the first report of a direct link between a sedimentological parameter and the composition and distribution of the sediment Beta-AOB and indicates the potential for using the Beta-AOB community composition in general and individual isolates or environmental clones in the Nitrosomonas oligotropha lineage in particular as bioindicators and biotracers of pollution or freshwater or wastewater input in coastal environments.Nitrification, the oxidation of ammonia to nitrate via nitrite, plays a critical role in the biogeochemical cycling of nitrogen and the formation of the large deep-sea nitrate reservoir (37, 46, 51). Because the N cycle may affect the global C cycle, shifts in N transformation processes may also affect the climate (30, 40). Nitrification is an important bioremediation process in human-perturbed estuarine and coastal ecosystems, where it may serve as a detoxification process for excess ammonia (14). If coupled to classical denitrification or anaerobic ammonium oxidation (anammox), these processes may remove most of the anthropogenic N pollution (51, 81). Bacterial nitrifiers may also cooxidize a variety of xenobiotic compounds (3, 49). On the other hand, nitrification may lead to enhanced production of the potent greenhouse gases nitric oxide (NO) and nitrous oxide (N₂O) (15). Because the input of excess ammonia stimulates the growth of ammonia-oxidizing microorganisms, research in coastal environments and ecosystems increasingly includes the study of microbial communities involved in nitrification.Marine nitrification is performed by chemolithoautotrophic proteobacteria and the newly discovered ammonia-oxidizing archaea (AOA) (47); nevertheless, reliable information on the individual contributions of each cohort to the process is still lacking (72) and the contributions likely vary in different environments (33, 51, 93). Beta- and gammaproteobacterial aerobic obligate ammonia-oxidizing bacteria (AOB) are known to catalyze the oxidization of ammonia to nitrite, the first and rate-limiting step of nitrification (3). Because of their monophyletic nature, diversity, and important environmental functionality, the betaproteobacterial AOB (Beta-AOB) have served as a model system in the study of fundamental questions in microbial ecology, including microbial community structure, distribution, activity, and environmental response (9, 49, 92).The growth of AOB is slow, and present isolates represent only a fraction of their natural diversity. Culture-independent molecular methods provide a more convenient and accurate approach for community analyses (76, 92). All AOB genomes contain at least one cluster of amoCAB genes encoding functional ammonia monooxygenase (AMO), which catalyzes the oxidation of ammonia to hydroxylamine (4). Because AmoA- and 16S rRNA-based phylogenies are congruent (73), the amoA gene has been extensively used as a molecular marker to explore and characterize the structure and diversity of AOB communities in a variety of estuarine and coastal environments (9, 10, 11, 13, 27, 32, 36, 43, 68, 88). Some of these studies indicated that local environmental factors such as salinity, pH, ammonium, and O₂ concentrations might be drivers for the formation of distinct AOB assemblages, in which individual lineages may have evolved differential ecophysiological adaptivity (4, 73, 85). Furthermore, differences in AmoA sequences may correlate with differences in isotopic discrimination during ammonia oxidation, implicating function-specific ammonia monooxygenases (15). Therefore, the AmoA sequences may provide information about the structure and composition of the AOB communities and their ecological function and response to environmental complexity and variability. Despite long-standing efforts, a complete understanding of these relationships is still lacking (9), especially in complex environments such as anthropogenic activity-impacted coastal areas.China consumes more than 20 million tons of N fertilizer each year, leading to a significant increase of coastal N pollution (38). Thus, the China coast is an important location for intense N biogeochemical cycling. Jiaozhou Bay is a large semienclosed water body of the temperate Yellow Sea in China. Eutrophication has become its most serious environmental problem, along with red tides, species loss, and contamination with toxic chemicals and harmful microbes (21, 23, 24, 28, 82, 91). In similar environments with a high input of nitrogenous compounds, surface sediment is a major site for nitrification due to a relatively high AOB abundance and activity (79).Although the China coast is important in N cycling and in related environmental and climatic issues, surprisingly very little is known, especially about the microbial processes and functions involved. On a global scale, it is currently not well understood how the AOB community structure, abundance, and distribution respond to coastal eutrophication, though partial knowledge is emerging (36, 49, 89). Recent studies indicated that spatial distribution and structure of the sediment AOA community could be influenced by a variety of environmental factors, of which continental input may play important roles in estuary and continental margin systems (22, 26). The sediment AOA community may serve as useful biotracers and bioindicators of specific environmental disturbance. Likewise, the sediment AOB community may also serve as biotracers or bioindicators of continental influence, such as eutrophication in coastal environments. In this study, the bacterial functional marker gene amoA was employed to test this hypothesis in the eutrophied Jiaozhou Bay.     

Distribution and Diversity of Archaeal and Bacterial Ammonia Oxidizers in Salt Marsh Sediments

Diversity and abundance of ammonia-oxidizing Betaproteobacteria (β-AOB) and archaea (AOA) were investigated in a New England salt marsh at sites dominated by short or tall Spartina alterniflora (SAS and SAT sites, respectively) or Spartina patens (SP site). AOA amoA gene richness was higher than β-AOB amoA richness at SAT and SP, but AOA and β-AOB richness were similar at SAS. β-AOB amoA clone libraries were composed exclusively of Nitrosospira-like amoA genes. AOA amoA genes at SAT and SP were equally distributed between the water column/sediment and soil/sediment clades, while AOA amoA sequences at SAS were primarily affiliated with the water column/sediment clade. At all three site types, AOA were always more abundant than β-AOB based on quantitative PCR of amoA genes. At some sites, we detected 10⁹ AOA amoA gene copies g of sediment⁻¹. Ratios of AOA to β-AOB varied over 2 orders of magnitude among sites and sampling dates. Nevertheless, abundances of AOA and β-AOB amoA genes were highly correlated. Abundance of 16S rRNA genes affiliated with Nitrosopumilus maritimus, Crenarchaeota group I.1b, and pSL12 were positively correlated with AOA amoA abundance, but ratios of amoA to 16S rRNA genes varied among sites. We also observed a significant effect of pH on AOA abundance and a significant salinity effect on both AOA and β-ΑΟΒ abundance. Our results expand the distribution of AOA to salt marshes, and the high numbers of AOA at some sites suggest that salt marsh sediments serve as an important habitat for AOA.Nitrification, the sequential oxidation of ammonia to nitrite and nitrate, is a critical step in the nitrogen cycle and is mediated by a suite of phylogenetically and physiologically distinct microorganisms. The recent discovery of ammonia oxidation among Archaea (17, 38) has led to a dramatic shift in the current model of nitrification and to new questions of niche differentiation between putative ammonia-oxidizing Archaea (AOA) and the more-well-studied ammonia-oxidizing Betaproteobacteria (β-AOB). Based on surveys of 16S rRNA genes and archaeal amoA genes, it is evident that AOA occupy a wide range of niches (10), suggesting a physiologically diverse group of Archaea. Additionally, in studies where AOA and β-AOB were both targeted, AOA were typically more abundant than their bacterial counterparts (19, 21, 42). However, there are reports of β-AOB outnumbering AOA in estuarine systems (6, 33), suggesting a possible shift in competitive dominance under certain conditions.Patterns of β-AOB diversity in estuaries have been well characterized and appear to be regulated by similar mechanisms within geographically disparate systems (4, 11, 32). However, AOA distribution and their role in nitrification relative to β-AOB remain to be determined. A few studies have begun to address this question in different estuaries, but no unifying patterns or mechanisms have emerged. Although β-AOB have been well studied along estuarine salinity gradients (1, 3, 4, 7, 11, 13, 22, 33, 39) and recent studies have begun to address AOA in estuaries (1, 6, 22, 32, 33), few have investigated β-AOB in salt marshes (9), and none has included AOA.In this study, we investigated the distribution and abundance of AOA and β-AOB based on the distribution and abundance of amoA genes in salt marsh sediments dominated by different types of vegetation. Although we equate the presence of archaeal amoA genes with the genetic potential to oxidize ammonia, we acknowledge the possibility that all Archaea that have amoA genes may not all represent functional ammonia oxidizers. Vegetation patterns of New England salt marshes are strongly correlated with marsh elevation and are controlled by a combination of interspecific competition and tolerance to physico-chemical stress (28). The dominant grasses of New England salt marshes are Spartina alterniflora and Spartina patens, which typically grow as pure stands. S. alterniflora is found in two phenotypically distinct but genetically identical forms, a tall and a short growth form (34). The tall S. alterniflora grows to heights of 1 to 2 m and is typically found at the edges of the marsh and along creek banks (SAT sites), while the short-form S. alterniflora may reach heights of only 30 cm and is found in sites (SAS sites) slightly higher on the marsh where soil drainage is limited and conditions are more reduced compared to SAT sites (14). Conversely, S. patens, due to its lower tolerance of salt and more reduced conditions, is found in sites (SP sites) highest on the marsh, in areas that receive less flooding (5). Because the marsh is subjected to daily tidal fluctuations, most sites experience periods of anoxia, the degree of which depends on the marsh elevation. We hypothesized that ammonia-oxidizing communities in areas dominated by different marsh grasses would reflect the different edaphic conditions associated with each type of grass, due to differences in vertical zonation in the marsh.     

Ammonia-Oxidizing Bacteria and Archaea in Groundwater Treatment and Drinking Water Distribution Systems

The ammonia-oxidizing prokaryote (AOP) community in three groundwater treatment plants and connected distribution systems was analyzed by quantitative real-time PCR and sequence analysis targeting the amoA gene of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Results demonstrated that AOB and AOA numbers increased during biological filtration of ammonia-rich anoxic groundwater, and AOP were responsible for ammonium removal during treatment. In one of the treatment trains at plant C, ammonia removal correlated significantly with AOA numbers but not with AOB numbers. Thus, AOA were responsible for ammonia removal in water treatment at one of the studied plants. Furthermore, an observed negative correlation between the dissolved organic carbon (DOC) concentration in the water and AOA numbers suggests that high DOC levels might reduce growth of AOA. AOP entered the distribution system in numbers ranging from 1.5 × 10³ to 6.5 × 10⁴ AOPs ml⁻¹. These numbers did not change during transport in the distribution system despite the absence of a disinfectant residual. Thus, inactive AOP biomass does not seem to be degraded by heterotrophic microorganisms in the distribution system. We conclude from our results that AOA can be commonly present in distribution systems and groundwater treatment, where they can be responsible for the removal of ammonia.Ammonia can be present in source water used for drinking water production or added to treated water with chlorine to form chloramines as a disinfectant. However, the presence of ammonia in drinking water is undesirable because nitrification might lead to toxic levels of nitrite (29) or adverse effects on water taste and odor (4) and might increase heterotrophic bacteria, including opportunistic pathogens (29). Two-thirds of the drinking water in The Netherlands is produced from groundwater. Most of the groundwater used for drinking water production is anoxic with relatively high concentrations of methane, iron, manganese, dissolved organic carbon (DOC), and ammonia. Treatment of anoxic groundwater aims at achieving biologically stable water, because drinking water in The Netherlands is distributed without a disinfectant residual. As a result, a highly efficient nitrification process during rapid medium filtration is required.Nitrification is the microbial oxidation of ammonia to nitrate and consists of two processes: the oxidation of ammonia to nitrite by ammonia-oxidizing prokaryotes (AOP) and the oxidation of nitrite to nitrate by nitrite-oxidizing bacteria (NOB). Recently it was shown that in addition to bacteria, archaea also are capable of ammonia oxidation (13). Since then, ammonia-oxidizing archaea (AOA) have been found in many different ecosystems, including wastewater treatment systems (10, 20, 24). However, it is currently unknown if AOA are present in drinking water treatment processes and distribution systems. Recent studies have focused on nitrification in drinking water treatment (16, 28). In those studies, AOB and NOB were enumerated by traditional most-probable-number (MPN) methods using selective liquid media. However, MPN methods are time-consuming and underestimate the numbers of AOP and NOB (3). Quantitative real-time PCR has been used to quantify AOB in drinking water (12) and might be a useful tool for quantifying AOB and AOA in drinking water.In our study, a real-time PCR method targeting the amoA gene of AOB or AOA was developed to quantify numbers of AOP in drinking water. This real-time PCR method was used together with a phylogenetic analysis of the amoA gene of AOB and AOA to do the following: (i) determine the treatment steps where AOP dominates in the groundwater treatment train of three drinking water production plants in The Netherlands, (ii) quantify the AOP entering the distribution system and determine the fate of AOP during transport in the distribution system, and (iii) elucidate the role of AOA in nitrification during drinking water treatment and in distribution systems.     

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).     

Vertical Distribution of Ammonia-Oxidizing Crenarchaeota and Methanogens in the Epipelagic Waters of Lake Kivu (Rwanda-Democratic Republic of the Congo)

Four stratified basins in Lake Kivu (Rwanda-Democratic Republic of the Congo) were sampled in March 2007 to investigate the abundance, distribution, and potential biogeochemical role of planktonic archaea. We used fluorescence in situ hybridization with catalyzed-reported deposition microscopic counts (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) fingerprinting, and quantitative PCR (qPCR) of signature genes for ammonia-oxidizing archaea (16S rRNA for marine Crenarchaeota group 1.1a [MCG1] and ammonia monooxygenase subunit A [amoA]). Abundance of archaea ranged from 1 to 4.5% of total DAPI (4′,6-diamidino-2-phenylindole) counts with maximal concentrations at the oxic-anoxic transition zone (∼50-m depth). Phylogenetic analysis of the archaeal planktonic community revealed a higher level of richness of crenarchaeal 16S rRNA gene sequences (21 of the 28 operational taxonomic units [OTUs] identified [75%]) over euryarchaeotal ones (7 OTUs). Sequences affiliated with the kingdom Euryarchaeota were mainly recovered from the anoxic water compartment and mostly grouped into methanogenic lineages (Methanosarcinales and Methanocellales). In turn, crenarchaeal phylotypes were recovered throughout the sampled epipelagic waters (0- to 100-m depth), with clear phylogenetic segregation along the transition from oxic to anoxic water masses. Thus, whereas in the anoxic hypolimnion crenarchaeotal OTUs were mainly assigned to the miscellaneous crenarchaeotic group, the OTUs from the oxic-anoxic transition and above belonged to Crenarchaeota groups 1.1a and 1.1b, two lineages containing most of the ammonia-oxidizing representatives known so far. The concomitant vertical distribution of both nitrite and nitrate maxima and the copy numbers of both MCG1 16S rRNA and amoA genes suggest the potential implication of Crenarchaeota in nitrification processes occurring in the epilimnetic waters of the lake.Lake Kivu is a meromictic lake located in the volcanic region between Rwanda and the Democratic Republic of the Congo and is the smallest of the African Great Rift Lakes. The monimolimnion of the lake contains a large amount of dissolved CO₂ and methane (300 km³ and 60 km³, respectively) as a result of geological and biological activity (24, 73, 85). This massive accumulation converts Lake Kivu into one of the largest methane reservoirs in the world and into a unique ecosystem for geomicrobiologists interested in the methane cycle and in risk assessment and management (34, 71, 72, 85). Comprehensive studies on the diversity and activity of planktonic populations of both large and small eukaryotes and their trophic interplay operating in the epilimnetic waters of the lake are available (33, 39, 49). Recent surveys have also provided a deeper insight into the seasonal variations of photosynthetic and heterotrophic picoplankton (67, 68), although very few data exist on the composition, diversity, and spatial distribution of bacterial and archaeal communities. In this regard, the studies conducted so far of the bacterial/archaeal ecology in Lake Kivu have been mostly focused on the implications on the methane cycle (34, 73), but none have addressed the presence and distribution of additional archaeal populations in the lake.During the last few years, microbial ecology studies carried out in a wide variety of habitats have provided compelling evidence of the ubiquity and abundance of mesophilic archaea (4, 10, 13, 19). Moreover, the discovery of genes encoding enzymes related to nitrification and denitrification in archaeal metagenomes from soil and marine waters (29, 86, 88) and the isolation of the first autotrophic archaeal nitrifier (40) demonstrated that some archaeal groups actively participate in the carbon and nitrogen cycles (56, 64, 69). In relation to aquatic environments, genetic markers of ammonia-oxidizing archaea (AOA) of the marine Crenarchaeota group 1.1a (MCG1) have consistently been found in water masses of several oceanic regions (6, 14, 17, 26, 28, 30, 37, 42, 51, 52, 89), estuaries (5, 9, 26, 53), coastal aquifers (26, 66), and stratified marine basins (15, 41, 44). Although less information is available for freshwater habitats, recent studies carried out in oligotrophic high-mountain and arctic lakes showed an important contribution of AOA in both the planktonic and the neustonic microbial assemblages (4, 61, 89).The oligotrophic nature of Lake Kivu and the presence of a well-defined redoxcline may provide an optimal niche for the development of autotrophic AOA populations. Unfortunately, no studies of the involvement of microbial planktonic populations in cycling nitrogen in the lake exist, and only data on the distribution of dissolved inorganic nitrogen species in relation to phytoplankton ecology (67, 68) and nutrient loading are available (54, 58). Our goals here were to ascertain whether or not archaeal populations other than methane-related lineages were relevant components of the planktonic microbial community and to determine whether the redox gradient imposed by the oxic-anoxic interphase acts as a threshold for their vertical distribution in epipelagic waters (0- to 100-m depth). To further explore the presence and potential activity of nitrifying archaeal populations in Lake Kivu, samples were analyzed for the abundance and vertical distribution of signature genes for these microorganisms, i.e., the 16S rRNA of MCG1 and the ammonia monooxygenase subunit A (amoA) gene by quantitative PCR.     

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.     

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.     

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.     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

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

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

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

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.