Impact of different in vitro electron donor/acceptor conditions on potential chemolithoautotrophic communities from marine pelagic redoxclines

Anaerobic or microaerophilic chemolithoautotrophic bacteria have been considered to be responsible for CO2 dark fixation in different pelagic redoxclines worldwide, but their involvement in redox processes is still not fully resolved. We investigated the impact of 17 different electron donor/acceptor combinations in water of pelagic redoxclines from the central Baltic Sea on the stimulation of bacterial CO2 dark fixation as well as on the development of chemolithoautotrophic populations. In situ, the highest CO2 dark fixation rates, ranging from 0.7 to 1.4 micromol liter(-1) day(-1), were measured directly below the redoxcline. In enrichment experiments, chemolithoautotrophic CO2 dark fixation was maximally stimulated by the addition of thiosulfate, reaching values of up to 9.7 micromol liter(-1) CO2 day(-1). Chemolithoautotrophic nitrate reduction proved to be an important process, with rates of up to 33.5 micromol liter(-1) NO3(-) day(-1). Reduction of Fe(III) or Mn(IV) was not detected; nevertheless, the presence of these potential electron acceptors influenced the development of stimulated microbial assemblages. Potential chemolithoautotrophic bacteria in the enrichment experiments were displayed on 16S ribosomal complementary DNA single-strand-conformation polymorphism fingerprints and identified by sequencing of excised bands. Sequences were closely related to chemolithoautotrophic Thiomicrospira psychrophila and Maorithyas hadalis gill symbiont (both Gammaproteobacteria) and to an uncultured nitrate-reducing Helicobacteraceae bacterium (Epsilonproteobacteria). Our data indicate that this Helicobacteraceae bacterium could be of general importance or even a key organism for autotrophic nitrate reduction in pelagic redoxclines.     

Epsilonproteobacteria Represent the Major Portion of Chemoautotrophic Bacteria in Sulfidic Waters of Pelagic Redoxclines of the Baltic and Black Seas

Recent studies have indicated that chemoautotrophic Epsilonproteobacteria might play an important role, especially as anaerobic or microaerophilic dark CO₂-fixing organisms, in marine pelagic redoxclines. However, knowledge of their distribution and abundance as actively CO₂-fixing microorganisms in pelagic redoxclines is still deficient. We determined the contribution of Epsilonproteobacteria to dark CO₂ fixation in the sulfidic areas of central Baltic Sea and Black Sea redoxclines by combining catalyzed reporter deposition-fluorescence in situ hybridization with microautoradiography using [¹⁴C]bicarbonate and compared it to the total prokaryotic chemoautotrophic activity. In absolute numbers, up to 3 × 10^{5 14}CO₂-fixing prokaryotic cells ml⁻¹ were enumerated in the redoxcline of the central Baltic Sea and up to 9 × 10^{4 14}CO₂-fixing cells ml⁻¹ were enumerated in the Black Sea redoxcline, corresponding to 29% and 12%, respectively, of total cell abundance. ¹⁴CO₂-incorporating cells belonged exclusively to the domain Bacteria. Among these, members of the Epsilonproteobacteria were approximately 70% of the cells in the central Baltic Sea and up to 100% in the Black Sea. For the Baltic Sea, the Sulfurimonas subgroup GD17, previously assumed to be involved in autotrophic denitrification, was the most dominant CO₂-fixing group. In conclusion, Epsilonproteobacteria were found to be mainly responsible for chemoautotrophic activity in the dark CO₂ fixation maxima of the Black Sea and central Baltic Sea redoxclines. These Epsilonproteobacteria might be relevant in similar habitats of the world's oceans, where high dark CO₂ fixation rates have been measured.     

Total Counts of Marine Bacteria Include a Large Fraction of Non-Nucleoid-Containing Bacteria (Ghosts)

Counts of heterotrophic bacteria in marine waters are usually in the order of 5 x 10(sup5) to 3 x 10(sup6) bacteria ml(sup-1). These numbers are derived from unspecific fluorescent staining techniques (J. E. Hobbie, R. J. Daley, and S. Jasper, Appl. Environ. Microbiol. 33:1225-1228, 1977; K. G. Porter and Y. S. Feig, Limnol. Oceanogr. 25:943-948, 1980) and are subsequently defined as total counts of bacteria. In samples from the Baltic Sea, the North Sea (Skagerrak), and the northeastern Mediterranean Sea, we found that only a minor fraction (2 to 32%) of total counts can be scored as bacteria with nucleoids. Lack of DNA no doubt means inactive cells; therefore, a much lower number of bacteria that grow at rates higher than those previously estimated must be responsible for the measured bacterial production in these seas. The remaining bacterium-sized and/or -shaped particles included in total counts may be cell residues of virus-lysed bacteria (ghosts) or remains of protozoan grazing.     

Impact of Different In Vitro Electron Donor/Acceptor Conditions on Potential Chemolithoautotrophic Communities from Marine Pelagic Redoxclines

Anaerobic or microaerophilic chemolithoautotrophic bacteria have been considered to be responsible for CO₂ dark fixation in different pelagic redoxclines worldwide, but their involvement in redox processes is still not fully resolved. We investigated the impact of 17 different electron donor/acceptor combinations in water of pelagic redoxclines from the central Baltic Sea on the stimulation of bacterial CO₂ dark fixation as well as on the development of chemolithoautotrophic populations. In situ, the highest CO₂ dark fixation rates, ranging from 0.7 to 1.4 μmol liter⁻¹ day⁻¹, were measured directly below the redoxcline. In enrichment experiments, chemolithoautotrophic CO₂ dark fixation was maximally stimulated by the addition of thiosulfate, reaching values of up to 9.7 μmol liter⁻¹ CO₂ day⁻¹. Chemolithoautotrophic nitrate reduction proved to be an important process, with rates of up to 33.5 μmol liter⁻¹ NO₃⁻ day⁻¹. Reduction of Fe(III) or Mn(IV) was not detected; nevertheless, the presence of these potential electron acceptors influenced the development of stimulated microbial assemblages. Potential chemolithoautotrophic bacteria in the enrichment experiments were displayed on 16S ribosomal complementary DNA single-strand-conformation polymorphism fingerprints and identified by sequencing of excised bands. Sequences were closely related to chemolithoautotrophic Thiomicrospira psychrophila and Maorithyas hadalis gill symbiont (both Gammaproteobacteria) and to an uncultured nitrate-reducing Helicobacteraceae bacterium (Epsilonproteobacteria). Our data indicate that this Helicobacteraceae bacterium could be of general importance or even a key organism for autotrophic nitrate reduction in pelagic redoxclines.     

Diversity and abundance of freshwater Actinobacteria along environmental gradients in the brackish northern Baltic Sea

Actinobacteria are highly abundant in pelagic freshwater habitats and also occur in estuarine environments such as the Baltic Sea. Because of gradients in salinity and other environmental variables estuaries offer natural systems for examining factors that determine Actinobacteria distribution. We studied abundance and community structure of Bacteria and Actinobacteria along two transects in the northern Baltic Sea. Quantitative (CARD-FISH) and qualitative (DGGE and clone libraries) analyses of community composition were compared with environmental parameters. Actinobacteria accounted for 22–27% of all bacteria and the abundance changed with temperature. Analysis of 549 actinobacterial 16S rRNA sequences from four clone libraries revealed a dominance of the freshwater clusters ac I and ac IV, and two new subclusters ( ac I-B scB-5 and ac IV-E) were assigned. Whereas ac I was present at all stations, occurrence of ac II and ac IV differed between stations and was related to dissolved organic carbon (DOC) and chlorophyll a (Chl a ) respectively. The prevalence of the ac I-A and ac I-B subclusters changed in relation to total phosphorus (Tot-P) and Chl a respectively. Community structure of Bacteria and Actinobacteria differed between the river station and all other stations, responding to differences in DOC, Chl a and bacterial production. In contrast, the composition of active Actinobacteria (analysis based on reversely transcribed RNA) changed in relation to salinity and Tot-P. Our study suggests an important ecological role of Actinobacteria in the brackish northern Baltic Sea. It highlights the need to address dynamics at the cluster or subcluster phylogenetic levels to gain insights into the factors regulating distribution and composition of Actinobacteria in aquatic environments.     

13C-isotope analyses reveal that chemolithoautotrophic Gamma- and Epsilonproteobacteria feed a microbial food web in a pelagic redoxcline of the central Baltic Sea

Marine pelagic redoxclines are zones of high dark CO₂ fixation rates, which can correspond up to 30% of the surface primary production. However, despite this significant contribution to the pelagic carbon cycle, the identity of most chemolithoautotrophic organisms is still unknown. Therefore, the aim of this study was to directly link the dark CO₂ fixation capacity of a pelagic redoxcline in the central Baltic Sea (Landsort Deep) with the identity of the main chemolithoautotrophs involved. Our approach was based on the analysis of natural carbon isotope signatures in fatty acid methyl esters (FAMEs) and on measurements of CO₂ incorporation in ¹³C-bicarbonate pulse experiments. The incorporation of ¹³C into chemolithoautotrophic cells was investigated by rRNA-based stable isotope probing (RNA-SIP) and FAME analysis after incubation for 24 and 72 h under in situ conditions. Our results demonstrated that fatty acids indicative of Proteobacteria were significantly enriched in ¹³C slightly below the chemocline. RNA-SIP analyses revealed that two different Gammaproteobacteria and three closely related Epsilonproteobacteria of the Sulfurimonas cluster were active dark CO₂-fixing microorganisms, with a time-dependent community shift between these groups. Labelling of Archaea was not detectable, but after 72 h of incubation the ¹³C-label had been transferred to a potentially bacterivorous ciliate related to Euplotes sp. Thus, RNA-SIP provided direct evidence for the contribution of chemolithoautotrophic production to the microbial food web in this marine pelagic redoxcline, emphasizing the importance of dark CO₂-fixing Proteobacteria within this habitat.     

The fabM gene product of Streptococcus mutans is responsible for the synthesis of monounsaturated fatty acids and is necessary for survival at low pH

Previously, it has been demonstrated that the membrane fatty acid composition of Streptococcus mutans is affected by growth pH (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004; R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189:89-92, 2000). Specifically, the proportion of monounsaturated fatty acids increases when the organism is grown in acidic environments; if the shift to increased monounsaturated fatty acids is blocked by the addition of a fatty acid biosynthesis inhibitor, the organism is rendered more acid sensitive (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004). Recently, work with Streptococcus pneumoniae has identified a novel enzyme, FabM, responsible for the production of monounsaturated fatty acids (H. Marrakchi, K. H. Choi, and C. O. Rock, J. Biol. Chem. 277:44809-44816, 2002). Using the published S. pneumoniae sequence, a putative FabM was identified in the S. mutans strain UA159. We generated a fabM strain that does not produce unsaturated fatty acids as determined by gas chromatography of fatty acid methyl esters. The mutant strain was extremely sensitive to low pH in comparison to the wild type; however, the acid-sensitive phenotype was relieved by growth in the presence of long-chain monounsaturated fatty acids or through genetic complementation. The strain exhibited reduced glycolytic capability and altered glucose-PTS activity. In addition, the altered membrane composition was more impermeable to protons and did not maintain a normal DeltapH. The results suggest that altered membrane composition can significantly affect the acid survival capabilities, as well as several enzymatic activities, of S. mutans.     

Identification of 16S ribosomal DNA-defined bacterial populations at a shallow submarine hydrothermal vent near Milos Island (Greece)

In a recent publication (S. M. Sievert, T. Brinkhoff, G. Muyzer, W. Ziebis, and J. Kuever, Appl. Environ. Microbiol. 65:3834-3842, 1999) we described spatiotemporal changes in the bacterial community structure at a shallow-water hydrothermal vent in the Aegean Sea near the isle of Milos (Greece). Here we describe identification and phylogenetic analysis of the predominant bacterial populations at the vent site and their distribution at the vent site as determined by sequencing of DNA molecules (bands) excised from denaturing gradient gels. A total of 36 bands could be sequenced, and there were representatives of eight major lineages of the domain Bacteria. Cytophaga-Flavobacterium and Acidobacterium were the most frequently retrieved bacterial groups. Less than 33% of the sequences exhibited 90% or more identity with cultivated organisms. The predominance of putative heterotrophic populations in the sequences retrieved is explained by the input of allochthonous organic matter at the vent site.     

Selective phylogenetic analysis targeting 16S rRNA genes of hyperthermophilic archaea in the deep-subsurface hot biosphere

International drilling projects for the study of microbial communities in the deep-subsurface hot biosphere have been expanded. Core samples obtained by deep drilling are commonly contaminated with mesophilic microorganisms in the drilling fluid, making it difficult to examine the microbial community by 16S rRNA gene clone library analysis. To eliminate mesophilic organism contamination, we previously developed a new method (selective phylogenetic analysis [SePA]) based on the strong correlation between the guanine-plus-cytosine (G+C) contents of the 16S rRNA genes and the optimal growth temperatures of prokaryotes, and we verified the method's effectiveness (H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006). In the present study we ascertained SePA's ability to eliminate contamination by archaeal rRNA genes, using deep-sea hydrothermal fluid (117 degrees C) and surface seawater (29.9 degrees C) as substitutes for deep-subsurface geothermal samples and drilling fluid, respectively. Archaeal 16S rRNA gene fragments, PCR amplified from the surface seawater, were denatured at 82 degrees C and completely digested with exonuclease I (Exo I), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84 degrees C because of their high G+C contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84 degrees C and digestion with Exo I completely eliminated archaeal 16S rRNA genes from the surface seawater. Our method was quite useful for culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.     

Identification of two proline transport systems in Staphylococcus aureus and their possible roles in osmoregulation.

The food-borne pathogen Staphylococcus aureus is distinguished from other food-borne pathogens by its ability to grow at water activity values below 0.90. Previous studies have indicated that proline accumulation mediated by transport represents a primary osmoregulatory strategy utilized by this bacterium (C. B. Anderson and L. D. Witter, Appl. Environ, Microbiol. 43:1501-1503, 1982; I. Koujima, H. Hayashi, K. Tomochika, A. Okabe, and Y. Kanemasa, Appl. Environ. Microbiol. 35:467-470, 1978; K. J. Miller, S. C. Zelt, and J.-H. Bae, Curr. Microbiol. 23:131-137, 1991). In this study, we demonstrate the presence of two proline transport systems within whole cells of S. aureus, a high-affinity transport system (Km, 7 microM) and a low-affinity transport system (Km, 420 microM). Our results indicate that the low-affinity proline transport system is osmotically activated and is the primary system responsible for the accumulation of proline by this pathogen during growth at low water activity.     

Identification of two proline transport systems in Staphylococcus aureus and their possible roles in osmoregulation.

The food-borne pathogen Staphylococcus aureus is distinguished from other food-borne pathogens by its ability to grow at water activity values below 0.90. Previous studies have indicated that proline accumulation mediated by transport represents a primary osmoregulatory strategy utilized by this bacterium (C. B. Anderson and L. D. Witter, Appl. Environ, Microbiol. 43:1501-1503, 1982; I. Koujima, H. Hayashi, K. Tomochika, A. Okabe, and Y. Kanemasa, Appl. Environ. Microbiol. 35:467-470, 1978; K. J. Miller, S. C. Zelt, and J.-H. Bae, Curr. Microbiol. 23:131-137, 1991). In this study, we demonstrate the presence of two proline transport systems within whole cells of S. aureus, a high-affinity transport system (Km, 7 microM) and a low-affinity transport system (Km, 420 microM). Our results indicate that the low-affinity proline transport system is osmotically activated and is the primary system responsible for the accumulation of proline by this pathogen during growth at low water activity.     

Comparison of enrichment broths for isolation of Campylobacter jejuni.

Growth of Campylobacter jejuni was compared in enrichment broths of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) and Park et al. (C. E. Park, Z. K. Stankiewicz, J. Lovett, and J. Hunt, Can. J. Microbiol. 27:841-842, 1981), as modified by Lovett et al. (J. Lovett, D. W. Francis, and J. M. Hunt, Appl. Environ. Microbiol. 46:459-462, 1983). Inoculated foods used were cream-pudding types, which may be cross-contaminated by improper handling, improper storage of meats prepared simultaneously, or the use of raw milk as an ingredient. Both broths adequately supported growth.     

Comparison of enrichment broths for isolation of Campylobacter jejuni

Growth of Campylobacter jejuni was compared in enrichment broths of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) and Park et al. (C. E. Park, Z. K. Stankiewicz, J. Lovett, and J. Hunt, Can. J. Microbiol. 27:841-842, 1981), as modified by Lovett et al. (J. Lovett, D. W. Francis, and J. M. Hunt, Appl. Environ. Microbiol. 46:459-462, 1983). Inoculated foods used were cream-pudding types, which may be cross-contaminated by improper handling, improper storage of meats prepared simultaneously, or the use of raw milk as an ingredient. Both broths adequately supported growth.     

Evidence for plasmid-mediated toxin and bacteriocin production in Clostridium botulinum type G.

A single 81-megadalton plasmid was previously isolated from each of six toxigenic strains of Clostridium botulinum type G (M. S. Strom, M. W. Eklund, and F. T. Poysky, Appl. Environ. Microbiol. 48:956-963, 1984). In this study, nontoxigenic derivatives isolated from each of the toxigenic strains following consecutive daily transfers in Trypticase (BBL Microbiology Systems, Cockeysville, Md.)-yeast extract-glucose broth at 44 degrees C simultaneously ceased to produce type G neurotoxin and to harbor the resident 81-megadalton plasmid. The nontoxigenic derivatives also ceased to produce bacteriocin and lost their immunity to the bacteriocin produced by the toxigenic strains. In contrast, all of the toxigenic isolates continued to carry the resident plasmid and to produce both bacteriocin and type G neurotoxin. This is the first evidence suggesting that the production of neurotoxin and bacteriocin by C. botulinum is mediated by a plasmid.     

Evidence for plasmid-mediated toxin and bacteriocin production in Clostridium botulinum type G

A single 81-megadalton plasmid was previously isolated from each of six toxigenic strains of Clostridium botulinum type G (M. S. Strom, M. W. Eklund, and F. T. Poysky, Appl. Environ. Microbiol. 48:956-963, 1984). In this study, nontoxigenic derivatives isolated from each of the toxigenic strains following consecutive daily transfers in Trypticase (BBL Microbiology Systems, Cockeysville, Md.)-yeast extract-glucose broth at 44 degrees C simultaneously ceased to produce type G neurotoxin and to harbor the resident 81-megadalton plasmid. The nontoxigenic derivatives also ceased to produce bacteriocin and lost their immunity to the bacteriocin produced by the toxigenic strains. In contrast, all of the toxigenic isolates continued to carry the resident plasmid and to produce both bacteriocin and type G neurotoxin. This is the first evidence suggesting that the production of neurotoxin and bacteriocin by C. botulinum is mediated by a plasmid.     

Identification of 16S Ribosomal DNA-Defined Bacterial Populations at a Shallow Submarine Hydrothermal Vent near Milos Island (Greece)

In a recent publication (S. M. Sievert, T. Brinkhoff, G. Muyzer, W. Ziebis, and J. Kuever, Appl. Environ. Microbiol. 65:3834–3842, 1999) we described spatiotemporal changes in the bacterial community structure at a shallow-water hydrothermal vent in the Aegean Sea near the isle of Milos (Greece). Here we describe identification and phylogenetic analysis of the predominant bacterial populations at the vent site and their distribution at the vent site as determined by sequencing of DNA molecules (bands) excised from denaturing gradient gels. A total of 36 bands could be sequenced, and there were representatives of eight major lineages of the domain Bacteria. Cytophaga-Flavobacterium and Acidobacterium were the most frequently retrieved bacterial groups. Less than 33% of the sequences exhibited 90% or more identity with cultivated organisms. The predominance of putative heterotrophic populations in the sequences retrieved is explained by the input of allochthonous organic matter at the vent site.     

Comparison of methods for isolating Campylobacter jejuni from raw milk.

The method of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) was compared with that of Lovett et al. (Appl. Environ. Microbiol. 46:459-462, 1983) for the ability to recover Campylobacter jejuni strains inoculated into raw milk at a concentration of less than 1 cell per g. The method of Lovett et al. gave significantly greater recovery proportions.     

Comparison of methods for isolating Campylobacter jejuni from raw milk

The method of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) was compared with that of Lovett et al. (Appl. Environ. Microbiol. 46:459-462, 1983) for the ability to recover Campylobacter jejuni strains inoculated into raw milk at a concentration of less than 1 cell per g. The method of Lovett et al. gave significantly greater recovery proportions.     

Heat inactivation of enteric viruses in dewatered wastewater sludge.

The effect of moisture content on the rates of heat inactivation of enteric viruses in wastewater sludge was determined. The protective effect of raw sludge on poliovirus previously observed (R. L. Ward, C. S. Ashley, and R. H. Moseley, Appl. Environ. Microbiol. 32:339--346, 1976) was found to be greatly enhanced in sludge dewatered by evaporation. Other enteroviruses responded in a similar fashion. This effect did not appear to be due merely to the state of dryness of the sludge samples because in humus-deficient soil, a relatively inert material, the rate of poliovirus inactivation by heat was not significantly altered through dewatering. Instead, this effect appeared to have been caused by protective substances in the sludge, such as detergents, which are concentrated through dewatering. As reported previously (R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol. 34:681-688, 1977; R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol 36:889--897, 1978) raw sludge is not protective of reovirus, but, instead, the ionic detergents in sludge cause the rate of heat inactivation of this virus to be accelerated. Dewatering of sludge, however, was found to partially reverse this virucidal effect. Evidence is presented indicating that this reversal is caused by an unidentified protective substance in sludge also concentrated through dewatering. Finally, it was shown that the effects of raw sludge on heat inactivation of poliovirus and reovirus are greatly reduced by composting, a result that correlated with the degradation of detergents.