Serpentinimonas gen. nov., Serpentinimonas raichei sp. nov., Serpentinimonas barnesii sp. nov. and Serpentinimonas maccroryi sp. nov., hyperalkaliphilic and facultative autotrophic bacteria isolated from terrestrial serpentinizing springs.

Three highly alkaliphilic bacterial strains designated as A1T, H1T and B1T were isolated from two highly alkaline springs at The Cedars, a terrestrial serpentinizing site. Cells from all strains were motile, Gram-negative and rod-shaped. Strains A1T, H1T and B1T were mesophilic (optimum, 30 °C), highly alkaliphilic (optimum, pH 11) and facultatively autotrophic. Major cellular fatty acids were saturated and monounsaturated hexadecenoic and octadecanoic acids. The genome size of strains A1T, H1T and B1T was 2 574 013, 2 475 906 and 2 623 236 bp, and the G+C content was 66.0, 66.2 and 66.1 mol%, respectively. Analysis of the 16S rRNA genes showed the highest similarity to the genera Malikia (95.1-96.4 %), Macromonas (93.0-93.6 %) and Hydrogenophaga (93.0-96.6 %) in the family Comamonadaceae. Phylogenetic analysis based on 16S rRNA gene and phylogenomic analysis based on core gene sequences revealed that the isolated strains diverged from the related species, forming a distinct branch. Average amino acid identity values of strains A1T, H1T and B1T against the genomes of related members in this family were below 67 %, which is below the suggested threshold for genera boundaries. Average nucleotide identity by blast values and digital DNA-DNA hybridization among the three strains were below 92.0 and 46.6 % respectively, which are below the suggested thresholds for species boundaries. Based on phylogenetic, genomic and phenotypic characterization, we propose Serpentinimonas gen. nov., Serpentinimonas raichei sp. nov. (type strain A1T=NBRC 111848T=DSM 103917T), Serpentinimonas barnesii sp. nov. (type strain H1T= NBRC 111849T=DSM 103920T) and Serpentinimonas maccroryi sp. nov. (type strain B1T=NBRC 111850T=DSM 103919T) belonging to the family Comamonadaceae. We have designated Serpentinimonas raichei the type species for the genus because it is the dominant species in The Cedars springs.

Anammox and beyond.

When looking back and wonder how we did it, I became even more aware of how my wanderings in microbiology are all linked, from the start of my PhD with Hans Veldkamp on sulphur-oxidizing bacteria in chemostats. My interests broadened from obligate chemolithoautotrophic bacteria to facultative organisms and the question about the ecological niches of these different metabolic types. The sulphide oxidizing bacteria also may be used to produce elemental sulphur, which can easily be removed from wastewater. This fitted in a long-standing collaboration with Dimitry Sorokin on the ecophysiology and application of alkaliphilic sulphur bacteria. Then came the denitrifying sulphur-oxidizing bacteria and their application to remove sulphide from wastewater, which lead to our interest in nitrate, nitrite and ammonium removal in general. The big surprise was the serendipitous discovery of the 'anammox'-process, whereby ammonium is anaerobically oxidized to dinitrogen gas with nitrite as electron acceptor. The early days of our anammox research are the main focus of this article, which describes the struggle of growing and identifying the most peculiar bacteria we ever came across. A specialized organelle, the anammoxosome was shown to be responsible for the key ammonium oxidation, whereby a rocket fuel, hydrazine, turned out to be an intermediate. Soon after we became aware that anammox is everywhere and in the marine environment makes up a major portion of the nitrogen cycle. The intense scientific collaboration with Mike Jetten and Mark van Loosdrecht and colleagues led to our further understanding and application of this fascinating process, which is briefly summarized in this article. My broader interest in environmental microbiology and microbial ecology has been a regularly returning theme, taking me all over the world to great collaborations lasting to this very day.

Growth yield and selection of nosZ clade II types in a continuous enrichment culture of N2 O respiring bacteria.

Nitrous oxide (N2 O) reducing microorganisms may be key in the mitigation of N2 O emissions from managed ecosystems. However, there is still no clear understanding of the physiological and bioenergetic implications of microorganisms possessing either of the two N2 O reductase genes (nosZ), clade I and the more recently described clade II type nosZ. It has been suggested that organisms with nosZ clade II have higher growth yields and a lower affinity constant (Ks ) for N2 O. We compared N2 O reducing communities with different nosZI/nosZII ratios selected in chemostat enrichment cultures, inoculated with activated sludge, fed with N2 O as a sole electron acceptor and growth limiting factor and acetate as electron donor. From the sequencing of the 16S rRNA gene, FISH and quantitative PCR of nosZ and nir genes, we concluded that betaproteobacterial denitrifying organisms dominated the enrichments with members within the family Rhodocyclaceae being highly abundant. When comparing cultures with different nosZI/nosZII ratios, we did not find support for (i) a more energy conserving N2 O respiration pathway in nosZ clade II systems, as reflected in the growth yield per mole of substrate, or (ii) a higher affinity for N2 O, defined by µmax /Ks , in organisms with nosZ clade II.

Life on N2O: deciphering the ecophysiology of N2O respiring bacterial communities in a continuous culture.

Reduction of the greenhouse gas N2O to N2 is a trait among denitrifying and non-denitrifying microorganisms having an N2O reductase, encoded by nosZ. The nosZ phylogeny has two major clades, I and II, and physiological differences among organisms within the clades may affect N2O emissions from ecosystems. To increase our understanding of the ecophysiology of N2O reducers, we determined the thermodynamic growth efficiency of N2O reduction and the selection of N2O reducers under N2O- or acetate-limiting conditions in a continuous culture enriched from a natural community with N2O as electron acceptor and acetate as electron donor. The biomass yields were higher during N2O limitation, irrespective of dilution rate and community composition. The former was corroborated in a continuous culture of Pseudomonas stutzeri and was potentially due to cytotoxic effects of surplus N2O. Denitrifiers were favored over non-denitrifying N2O reducers under all conditions and Proteobacteria harboring clade I nosZ dominated. The abundance of nosZ clade II increased when allowing for lower growth rates, but bacteria with nosZ clade I had a higher affinity for N2O, as defined by µmax/Ks. Thus, the specific growth rate is likely a key factor determining the composition of communities living on N2O respiration under growth-limited conditions.

Exploring microbial N2 O reduction: a continuous enrichment in nitrogen free medium.

N2 O is a potent greenhouse gas, but also a potent electron acceptor. In search of thermodynamically favourable - yet undescribed - metabolic pathways involving N2 O reduction, we set up a continuous microbial enrichment, inoculated with activated sludge, fed with N2 O as the sole electron acceptor and acetate as an electron donor. A nitrogen-free mineral medium was used with the intention of creating a selective pressure towards organisms that would use N2 O directly as source of nitrogen for cell synthesis. Instead, we obtained a culture dominated by microorganisms of the Rhodocyclaceae family growing by N2 O reduction to N2 coupled to N2 fixation. Biomass yields of this culture were 40% lower than those of a previously reported culture grown under comparable conditions but with an NH4+-amended medium, as expected from the extra energy expense of N2 fixation. Interestingly, we found no significant difference in yields whether N2 O or acetate was the growth-limiting substrate in the chemostat in contrast to the study with NH4+-amended medium, in which biomass yields were roughly 30% lower during acetate limiting conditions.

Fermentative Bacteria Influence the Competition between Denitrifiers and DNRA Bacteria.

Denitrification and dissimilatory reduction to ammonium (DNRA) are competing nitrate-reduction processes that entail important biogeochemical consequences for nitrogen retention/removal in natural and man-made ecosystems. The nature of the available carbon source and electron donor have been suggested to play an important role on the outcome of this microbial competition. In this study, the influence of lactate as fermentable carbon source on the competition for nitrate was investigated for varying ratios of lactate and nitrate in the influent (Lac/N ratio). The study was conducted in an open chemostat culture, enriched from activated sludge, under strict anoxia. The mechanistic explanation of the conversions observed was based on integration of results from specific batch tests with biomass from the chemostat, molecular analysis of the biomass enriched, and a computational model. At high Lac/N ratio (2.97 mol/mol) both fermentative and respiratory nitrate reduction to ammonium occurred, coupled to partial oxidation of lactate to acetate, and to acetate oxidation respectively. Remaining lactate was fermented to propionate and acetate. At a decreased Lac/N ratio (1.15 mol/mol), the molar percentage of nitrate reduced to ammonium decreased to 58%, even though lactate was supplied in adequate amounts for full ammonification and nitrate remained the growth limiting compound. Data evaluation at this Lac/N ratio suggested conversions were comparable to the higher Lac/N ratio, except for lactate oxidation to acetate that was coupled to denitrification instead of ammonification. Respiratory DNRA on acetate was likely catalyzed by two Geobacter species related to G. luticola and G. lovleyi. Two Clostridiales members were likely responsible for lactate fermentation and partial lactate fermentation to acetate coupled to fermentative DNRA. An organism related to Propionivibrio militaris was identified as the organism likely responsible for denitrification. The results of this study clearly show that not only the ratio of available substrates, but also the nature of the electron donor influences the outcome of competition between DNRA and denitrification. Apparently, fermentative bacteria are competitive for the electron donor and thereby alter the ratio of available substrates for nitrate reduction.

Unusual metabolic diversity of hyperalkaliphilic microbial communities associated with subterranean serpentinization at The Cedars.

Water from The Cedars springs that discharge from serpentinized ultramafic rocks feature highly basic (pH=~12), highly reducing (Eh<-550 mV) conditions with low ionic concentrations. These conditions make the springs exceptionally challenging for life. Here, we report the metagenomic data and recovered draft genomes from two different springs, GPS1 and BS5. GPS1, which was fed solely by a deep groundwater source within the serpentinizing system, was dominated by several bacterial taxa from the phyla OD1 ('Parcubacteria') and Chloroflexi. Members of the GPS1 community had, for the most part, the smallest genomes reported for their respective taxa, and encoded only archaeal (A-type) ATP synthases or no ATP synthases at all. Furthermore, none of the members encoded respiration-related genes and some of the members also did not encode key biosynthesis-related genes. In contrast, BS5, fed by shallow water, appears to have a community driven by hydrogen metabolism and was dominated by a diverse group of Proteobacteria similar to those seen in many terrestrial serpentinization sites. Our findings indicated that the harsh ultrabasic geological setting supported unexpectedly diverse microbial metabolic strategies and that the deep-water-fed springs supported a community that was remarkable in its unusual metagenomic and genomic constitution.

Role of nitrite in the competition between denitrification and DNRA in a chemostat enrichment culture.

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are two microbial processes that compete for oxidized nitrogen compounds in the environment. The objective of this work was to determine the role of nitrite versus nitrate as terminal electron acceptor on the competition between DNRA and denitrification. Initially, a mixed culture chemostat was operated under nitrate limitation and performed DNRA. Stepwise, the influent nitrate was replaced with nitrite until nitrite was the sole electron acceptor and N-source present. Despite changing the electron acceptor from nitrate to nitrite, the dominant process remained DNRA and the same dominant organism closely related to Geobacter lovleyi was identified. Contrary to previous studies conducted with a complex substrate in marine microbial communities, the conclusion of this work is that nitrate versus nitrite as electron acceptor does not generally control the competition between DNRA and denitrification. Our results show that the effect of this ratio must be interpreted in combination with other environmental factors, such as the type and complexity of the electron donor, pH, or sulfide concentrations.

DNRA and Denitrification Coexist over a Broad Range of Acetate/N-NO3- Ratios, in a Chemostat Enrichment Culture.

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) compete for nitrate in natural and engineered environments. A known important factor in this microbial competition is the ratio of available electron donor and elector acceptor, here expressed as Ac/N ratio (acetate/nitrate-nitrogen). We studied the impact of the Ac/N ratio on the nitrate reduction pathways in chemostat enrichment cultures, grown on acetate mineral medium. Stepwise, conditions were changed from nitrate limitation to nitrate excess in the system by applying a variable Ac/N ratio in the feed. We observed a clear correlation between Ac/N ratio and DNRA activity and the DNRA population in our reactor. The DNRA bacteria dominated under nitrate limiting conditions in the reactor and were outcompeted by denitrifiers under limitation of acetate. Interestingly, in a broad range of Ac/N ratios a dual limitation of acetate and nitrate occurred with co-occurrence of DNRA bacteria and denitrifiers. To explain these observations, the system was described using a kinetic model. The model illustrates that the Ac/N effect and concomitant broad dual limitation range related to the difference in stoichiometry between both processes, as well as the differences in electron donor and acceptor affinities. Population analysis showed that the presumed DRNA-performing bacteria were the same under nitrate limitation and under dual limiting conditions, whereas the presumed denitrifying population changed under single and dual limitation conditions.

Physiological and genomic features of highly alkaliphilic hydrogen-utilizing Betaproteobacteria from a continental serpentinizing site.

Serpentinization, or the aqueous alteration of ultramafic rocks, results in challenging environments for life in continental sites due to the combination of extremely high pH, low salinity and lack of obvious electron acceptors and carbon sources. Nevertheless, certain Betaproteobacteria have been frequently observed in such environments. Here we describe physiological and genomic features of three related Betaproteobacterial strains isolated from highly alkaline (pH 11.6) serpentinizing springs at The Cedars, California. All three strains are obligate alkaliphiles with an optimum for growth at pH 11 and are capable of autotrophic growth with hydrogen, calcium carbonate and oxygen. The three strains exhibit differences, however, regarding the utilization of organic carbon and electron acceptors. Their global distribution and physiological, genomic and transcriptomic characteristics indicate that the strains are adapted to the alkaline and calcium-rich environments represented by the terrestrial serpentinizing ecosystems. We propose placing these strains in a new genus 'Serpentinomonas'.

Microbial diversity in The Cedars, an ultrabasic, ultrareducing, and low salinity serpentinizing ecosystem.

The Cedars, in coastal northern California, is an active site of peridotite serpentinization. The spring waters that emerge from this system feature very high pH, low redox potential, and low ionic concentrations, making it an exceptionally challenging environment for life. We report a multiyear, culture-independent geomicrobiological study of three springs at The Cedars that differ with respect to the nature of the groundwater feeding them. Within each spring, both geochemical properties and microbial diversity in all three domains of life remained stable over a 3-y period, with multiple samples each year. Between the three springs, however, the microbial communities showed considerable differences that were strongly correlated with the source of the serpentinizing groundwater. In the spring fed solely by deep groundwater, phylum Chloroflexi, class Clostridia, and candidate division OD1 were the major taxa with one phylotype in Euryarchaeota. Less-abundant phylotypes include several minor members from other candidate divisions and one phylotype that was an outlier of candidate division OP3. In the springs fed by the mixture of deep and shallow groundwater, organisms close to the Hydrogenophaga within Betaproteobacteria dominated and coexisted with the deep groundwater community members. The shallow groundwater community thus appears to be similar to those described in other terrestrial serpentinizing sites, whereas the deep community is distinctly different from any other previously described terrestrial serpentinizing community. These unique communities have the potential to yield important insights into the development and survival of life in these early-earth analog environments.

Mariprofundus ferrooxydans PV-1 the first genome of a marine Fe(II) oxidizing Zetaproteobacterium.

Mariprofundus ferrooxydans PV-1 has provided the first genome of the recently discovered Zetaproteobacteria subdivision. Genome analysis reveals a complete TCA cycle, the ability to fix CO(2), carbon-storage proteins and a sugar phosphotransferase system (PTS). The latter could facilitate the transport of carbohydrates across the cell membrane and possibly aid in stalk formation, a matrix composed of exopolymers and/or exopolysaccharides, which is used to store oxidized iron minerals outside the cell. Two-component signal transduction system genes, including histidine kinases, GGDEF domain genes, and response regulators containing CheY-like receivers, are abundant and widely distributed across the genome. Most of these are located in close proximity to genes required for cell division, phosphate uptake and transport, exopolymer and heavy metal secretion, flagellar biosynthesis and pilus assembly suggesting that these functions are highly regulated. Similar to many other motile, microaerophilic bacteria, genes encoding aerotaxis as well as antioxidant functionality (e.g., superoxide dismutases and peroxidases) are predicted to sense and respond to oxygen gradients, as would be required to maintain cellular redox balance in the specialized habitat where M. ferrooxydans resides. Comparative genomics with other Fe(II) oxidizing bacteria residing in freshwater and marine environments revealed similar content, synteny, and amino acid similarity of coding sequences potentially involved in Fe(II) oxidation, signal transduction and response regulation, oxygen sensation and detoxification, and heavy metal resistance. This study has provided novel insights into the molecular nature of Zetaproteobacteria.

Segregation of biomass in cyclic anaerobic/aerobic granular sludge allows the enrichment of anaerobic ammonium oxidizing bacteria at low temperatures.

A cyclic anaerobic/aerobic bubble column reactor was run for 420 days to study the competition for nitrite between nitrite oxidizing bacteria (NOB) and anaerobic ammonium oxidizing bacteria (Anammox) at low temperatures. An anaerobic feeding period with nitrite and ammonium in the influent followed by an aerated period was applied resulting in a biomass specific conversion rate of 0.18 ± 0.02 [gN(2) - N · gVSS(-1)· day(-1)] when the dissolved oxygen concentration was maintained at 1.0 mgO(2) · L(-1). An increase in white granules was observed in the reactor which were mainly located at the top of the settled sludge bed, whereas red granules were located at the bottom. FISH, activity tests, and qPCR techniques revealed that red biomass was dominated by Anammox bacteria and white granules by NOB. Granules from the top of the sludge bed were smaller and therefore had a higher aerobic volume fraction, a lower density, and consequently a slower settling rate. Sludge was manually removed from the top of the settled sludge bed to selectively remove NOB which resulted in an increased overall biomass specific N-conversion rate of 0.32 ± 0.02 [gN(2) - N · gVSS(-1) · day(-1)]. Biomass segregation in granular sludge reactors gives an extra opportunity to select for specific microbial groups by applying a different SRT for different microbial groups.

The microbial sulfur cycle at extremely haloalkaline conditions of soda lakes.

Soda lakes represent a unique ecosystem with extremely high pH (up to 11) and salinity (up to saturation) due to the presence of high concentrations of sodium carbonate in brines. Despite these double extreme conditions, most of the lakes are highly productive and contain a fully functional microbial system. The microbial sulfur cycle is among the most active in soda lakes. One of the explanations for that is high-energy efficiency of dissimilatory conversions of inorganic sulfur compounds, both oxidative and reductive, sufficient to cope with costly life at double extreme conditions. The oxidative part of the sulfur cycle is driven by chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacteria (SOB), which are unique for soda lakes. The haloalkaliphilic SOB are present in the surface sediment layer of various soda lakes at high numbers of up to 10(6) viable cells/cm(3). The culturable forms are so far represented by four novel genera within the Gammaproteobacteria, including the genera Thioalkalivibrio, Thioalkalimicrobium, Thioalkalispira, and Thioalkalibacter. The latter two were only found occasionally and each includes a single species, while the former two are widely distributed in various soda lakes over the world. The genus Thioalkalivibrio is the most physiologically diverse and covers the whole spectrum of salt/pH conditions present in soda lakes. Most importantly, the dominant subgroup of this genus is able to grow in saturated soda brines containing 4 M total Na(+) - a so far unique property for any known aerobic chemolithoautotroph. Furthermore, some species can use thiocyanate as a sole energy source and three out of nine species can grow anaerobically with nitrogen oxides as electron acceptor. The reductive part of the sulfur cycle is active in the anoxic layers of the sediments of soda lakes. The in situ measurements of sulfate reduction rates and laboratory experiments with sediment slurries using sulfate, thiosulfate, or elemental sulfur as electron acceptors demonstrated relatively high sulfate reduction rates only hampered by salt-saturated conditions. However, the highest rates of sulfidogenesis were observed not with sulfate, but with elemental sulfur followed by thiosulfate. Formate, but not hydrogen, was the most efficient electron donor with all three sulfur electron acceptors, while acetate was only utilized as an electron donor under sulfur-reducing conditions. The native sulfidogenic populations of soda lakes showed a typical obligately alkaliphilic pH response, which corresponded well to the in situ pH conditions. Microbiological analysis indicated a domination of three groups of haloalkaliphilic autotrophic sulfate-reducing bacteria belonging to the order Desulfovibrionales (genera Desulfonatronovibrio, Desulfonatronum, and Desulfonatronospira) with a clear tendency to grow by thiosulfate disproportionation in the absence of external electron donor even at salt-saturating conditions. Few novel representatives of the order Desulfobacterales capable of heterotrophic growth with volatile fatty acids and alcohols at high pH and moderate salinity have also been found, while acetate oxidation was a function of a specialized group of haloalkaliphilic sulfur-reducing bacteria, which belong to the phylum Chrysiogenetes.

Aerobic carboxydotrophy under extremely haloalkaline conditions in Alkalispirillum/Alkalilimnicola strains isolated from soda lakes.

Aerobic enrichments from soda lake sediments with CO as the only substrate resulted in the isolation of five bacterial strains capable of autotrophic growth with CO at extremely high pH and salinity. The strains belonged to the Alkalispirillum/Alkalilimnicola cluster in the Gammaproteobacteria, where the ability to oxidize CO, but not growth with CO, has been demonstrated previously. The growth with CO was possible only at an oxygen concentration below 5 % and CO concentration below 20 % in the gas phase. The isolates were also capable of growth with formate but not with H(2). The carboxydotrophic growth occurred within a narrow pH range from 8 to 10.5 (optimum at 9.5) and a broad salt concentration from 0.3 to 3.5 M total Na(+) (optimum at 1.0 M). Cells grown on CO had high respiration activity with CO and formate, while the cells grown on formate actively oxidized formate alone. In CO-grown cells, CO-dehydrogenase (CODH) activity was detectable both in soluble and membrane fractions, while the NAD-independent formate dehydrogenase (FDH) resided solely in membranes. The results of total protein profiling and the failure to detect CODH with conventional primers for the coxL gene indicated that the CO-oxidizing enzyme in haloalkaliphilic isolates might differ from the classical aerobic CODH complex. A single cbbL gene encoding the RuBisCO large subunit was detected in all strains, suggesting the presence of the Calvin cycle of inorganic carbon fixation. Overall, these results demonstrated the possibility of aerobic carboxydotrophy under extremely haloalkaline conditions.

Physiology and behaviour of marine Thioploca.

Among prokaryotes, the large vacuolated marine sulphur bacteria are unique in their ability to store, transport and metabolize significant quantities of sulphur, nitrogen, phosphorus and carbon compounds. In this study, unresolved questions of metabolism, storage management and behaviour were addressed in laboratory experiments with Thioploca species collected on the continental shelf off Chile. The Thioploca cells had an aerobic metabolism with a potential oxygen uptake rate of 1760 micromol O2 per dm(3) biovolume per h, equivalent to 4.4 nmol O2 per min per mg protein. When high ambient sulphide concentrations (approximately 200 microM) were present, a sulphide uptake of 6220+/-2230 micromol H2S per dm(3) per h, (mean+/-s.e.m., n=4) was measured. This sulphide uptake rate was six times higher than the oxidation rate of elemental sulphur by oxygen or nitrate, thus indicating a rapid sulphur accumulation by Thioploca. Thioploca reduce nitrate to ammonium and we found that dinitrogen was not produced, neither through denitrification nor through anammox activity. Unexpectedly, polyphosphate storage was not detectable by microautoradiography in physiological assays or by staining and microscopy. Carbon dioxide fixation increased when nitrate and nitrite were externally available and when organic carbon was added to incubations. Sulphide addition did not increase carbon dioxide fixation, indicating that Thioploca use excess of sulphide to rapidly accumulate sulphur rather than to accelerate growth. This is interpreted as an adaptation to infrequent high sulphate reduction rates in the seabed. The physiology and behaviour of Thioploca are summarized and the adaptations to an environment, dominated by infrequent oxygen availability and periods of high sulphide abundance, are discussed.

Thiohalorhabdus denitrificans gen. nov., sp. nov., an extremely halophilic, sulfur-oxidizing, deep-lineage gammaproteobacterium from hypersaline habitats.

Seven strains of extremely halophilic and obligately chemolithoautotrophic sulfur-oxidizing bacteria (SOB) were enriched and isolated at 4 M NaCl from sediments of hypersaline inland lakes in south-eastern Siberia and a Mediterranean sea solar saltern. Cells of the novel isolates were spindle-like, long and non-motile rods with a Gram-negative type of cell wall. They were obligately chemolithoautotrophic SOB using thiosulfate and tetrathionate as electron donors and represent the first example of extremely halophilic chemolithoautotrophs that are able to grow anaerobically with nitrate as electron acceptor. The characteristic feature of the group was the production of large amounts of tetrathionate as an intermediate during the oxidation of thiosulfate to sulfate. With thiosulfate, the novel strains grew within the pH range from 6.5 to 8.2 (optimum at pH 7.5-7.8) and at NaCl concentrations from 1.5 to 4.0 M (optimum at 3.0 M). Cells grown at 4 M NaCl accumulated extremely high concentrations of glycine betaine as a compatible solute. The dominant cellular fatty acids were 10MeC(16 : 0) and C(16 : 0). Based on the DNA-DNA relatedness values, the isolates consisted of a single genomic species and had a similar phenotype. Phylogenetic analysis placed the novel bacteria in the class Gammaproteobacteria as an independent lineage with no significant relationship to any other genera in this class. On the basis of phenotypic and genotypic analysis, the group is proposed to represent a new genus, Thiohalorhabdus gen. nov., with Thiohalorhabdus denitrificans gen. nov., sp. nov. as the type species (type strain HL 19(T)=DSM 15699(T)=UNIQEM U223(T)).

Expression of copA and cusA in Shewanella during copper stress.

Copper homeostasis is tightly regulated in all living cells as a result of the necessity and toxicity of this metal in free cationic form. In Gram-negative bacteria CPx-type ATPases (e.g. CopA in Escherichia coli) and heavy-metal efflux RND proteins (e.g. CusA in E. coli) play an important role in transport of copper across the cytoplasmic and outer membrane. We investigated the expression of CusA- and CopA-like proteins in Shewanella oneidensis MR1 and Shewanella strain MB4, a Mn(IV)-reducing isolate from a metal-polluted harbour sediment. Q-PCR analysis of total mRNA extracted from cultures grown under aerobic conditions with 25 microM copper showed significantly increased expression of cusA (Student's t-test: MR1, P<0.0001; MB4, P=0.0006). This gene was also induced in the presence of 100 microM copper and 10 or 25 microM cadmium in both tested strains. In the absence of oxygen, with fumarate as final electron acceptor and 100 microM copper, a prolonged lag phase (5 h) was observed and general fitness decreased as evidenced by twofold lower copy numbers of 16S rRNA compared to aerobic conditions. cusA expression in cells grown under these conditions remained at comparable levels (MR1) or was slightly decreased (MB4), compared to aerobic copper challenges. A gene homologous to the copA gene of S. oneidensis was not detected in strain MB4. Although low copA copy numbers were observed in strain MR1 under conditions with 25 and 100 microM copper, copA was not detected in mRNA from cultures grown in the presence of 10 microM cadmium, or in the absence of added heavy metals. However, copA was highly induced under anaerobic conditions with 100 microM copper (P=0.0011). These results suggest essentially different roles for the two proteins CopA and CusA in the copper response in S. oneidensis MR1, similar to findings in more metal-resistant bacteria such as Escherichia coli and Cupriavidus metallidurans.

Anammox bacteria: from discovery to application.

Anaerobic ammonium oxidation (anammox) bacteria, which were discovered in waste-water sludge in the early 1990s, have the unique metabolic ability to combine ammonium and nitrite or nitrate to form nitrogen gas. This discovery led to the realization that a substantial part of the enormous nitrogen losses that are observed in the marine environment--up to 50% of the total nitrogen turnover--were due to the activity of these bacteria. In this Timeline, Gijs Kuenen recalls the discovery of these unique microorganisms and describes the continuing elucidation of their roles in environmental and industrial microbiology.

Influence of salts and pH on growth and activity of a novel facultatively alkaliphilic, extremely salt-tolerant, obligately chemolithoautotrophic sufur-oxidizing Gammaproteobacterium Thioalkalibacter halophilus gen. nov., sp. nov. from South-Western Siberian soda lakes.

A chemolithoautotrophic sulfur-oxidizing bacterium (SOB) strain ALCO 1 capable of growing at both near-neutral and extremely alkaline pH was isolated from hypersaline soda lakes in S-W Siberia (Altai, Russia). Strain ALCO 1 represents a novel separate branch within the halothiobacilli in the Gammaproteobacteria, which, so far, contained only neutro-halophilic SOB. On the basis of its unique phenotypic properties and distant phylogeny, strain ALCO 1 is proposed as a new genus and species Thioalkalibacter halophilus gen. nov. sp. nov. ALCO 1 was able to grow within a broad range of salinity (0.5-3.5 M of total sodium) with an optimum at around 1 M Na+, and pH (7.2-10.2, pHopt at around 8.5). Na+ was required for sulfur-dependent respiration in ALCO 1. The neutral (NaCl)-grown chemostat culture had a much lower maximum growth rate (micromax), respiratory activity and total cytochrome c content than its alkaline-grown counterpart. The specific concentration of osmolytes (ectoine and glycine-betaine) produced at neutral pH and 3 M NaCl was roughly two times higher than at pH 10 in soda. Altogether, strain ALCO 1 represents an interesting chemolithoautotrophic model organism for comparative investigations of bacterial adaptations to high salinity and pH.