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PubMed2000

Identification of major subgroups of ammonia-oxidizing bacteria in environmental samples by T-RFLP analysis of amoA PCR products.

A cloning-independent method based on T-RFLP (terminal restriction fragment length polymorphism) analysis of amoA PCR products was developed to identify major subgroups of autotrophic ammonia oxidizers of the beta-subclass of the class Proteobacteria in total community DNA. Based on a database of 28 partial gene sequences encoding the active-site polypeptide of ammonia monooxygenase (amoA), defined lengths of terminal restriction fragments (= operational taxonomic units, OTUs) of amoA were predicted to correlate in TaqI-based T-RFLP analysis with phylogenetically defined subgroups of ammonia oxidizers. Members of the genus Nitrosospira showed a specific OTU of 283 bp in length, while a fragment size of 219 bp was indicative of Nitrosomonas-like sequence types including N. europaea, N. eutropha, and N. halophila. Two amoA sequence clusters designated previously as the lineages 'PluBsee' and 'Schöhsee' [Rotthauwe, J.-H., Witzel, K.-P., Liesack, W., 1997. Appl. Environ. Microbiol. 63, 4704-4712] shared a TaqI-based OTU with a fragment size of 48 bp, but sequence types of these two lineages could be differentiated by AluI-based T-RFLP analysis. A survey of various environmental samples and enrichment cultures by T-RFLP analysis and by comparative analysis of cloned amoA sequences confirmed the predicted correlations between distinct OTUs and phylogenetic information. Our data suggest that amoA-based T-RFLP analysis is a reliable tool to rapidly assess the complexity of ammonia-oxidizing communities in environmental samples with respect to the presence of major subgroups, i.e. nitrosospiras versus nitrosomonads.

Ammonia↗

PubMed2002

MELISSA: a loop of interconnected bioreactors to develop life support in space.

The development of a loop of interconnected continuous bioreactors, aimed to provide life support in space, is reported. The complete loop concept consists of four bioreactors and one higher plant compartment. For its realization the continuous and controlled operation of the bioreactors is characterized, up to the pilot scale level, first for each individual reactor, second for the interconnected reactor operation. The results obtained with the two more advanced bioreactors in the Micro Ecological Life Support System Alternative (MELISSA) loop are described more specifically. These reactors consist of a packed-bed reactor working with an immobilized co-culture of Nitrosomonas and Nitrobacter cells, and an external loop gas-lift photobioreactor for the culture of the cyanobacteria Spirulina platensis. Their individual operation for long duration runs has been achieved and characterized, and their interconnected operation at pilot scale is reported.

Bioreactors↗

PubMed2001

Soft particle analysis of bacterial cells and its interpretation of cell adhesion behaviors in terms of DLVO theory.

The electrokinetic properties of two nitrifying strains, Nitrosomonas europaea and Nitrobacter winogradskyi, and three heterotrophic bacteria, Escherichia coli, Pseudomonas putida and Pseudomonas aeruginosa, were examined by electrophoretic mobility measurement and analyzed using the soft particle electrophoresis theory that is suitable for biological particles. The bacterial adhesion characteristics onto glass bead substratum were also evaluated by packed bed method. The mobility of the bacterial cells employed converged to a non-zero value as the ionic concentration increased, suggesting that the bacterial cells exhibited typical soft particle characteristics. Moreover, cell surface potentials based on the soft particle theory were lower than those estimated by the conventional Smoluchowski formula, i.e. zeta potential. Cell collision efficiencies onto glass beads (alpha(0)) were largely dependent on interfacial interaction, although almost electrically neutral P. aeruginosa did not follow that trend. From a comparison of alpha(0) with DLVO interaction energy maximum (V(max)), it was assumed that heterocoagulation between cell and substratum at primary minimum potential took place under V(max) of 24-34 kT based on soft particle analysis. On the other hand, V(max) predictions using the Smoluchowski theory gave 81-223 kT, which indicated the possibility of overestimating electrostatic repulsive forces by the conventional Smoluchowski theory. Thus, the application of this new electrophoresis theory to several kinds of bacterial cells has led to the revision of the interpretation of bacterial mobility data and provided a more detailed understanding of the bacterial adhesion phenomenon.

Journal Article↗

PubMed2004

Start-up of autotrophic nitrogen removal reactors via sequential biocatalyst addition.

A procedure for start-up of oxygen-limited autotrophic nitrification-denitrification (OLAND) in a lab-scale rotating biological contactor (RBC) is presented. In this one-step process, NH4+ is directly converted to N2 without the need for an organic carbon source. The approach is based on a sequential addition of two types of easily available biocatalyst to the reactor during start-up: aerobic nitrifying and anaerobic, granular methanogenic sludge. The first is added as a source of aerobic ammonia-oxidizing bacteria (AAOB), the second as a possible source of planctomycetes including anaerobic ammonia-oxidizing bacteria (AnAOB). The initial nitrifying biofilm serves as a matrix for anaerobic cell incorporation. By subsequently imposing oxygen limitation, one can create an optimal environment for autotrophic N removal. In this way, N removal of about 250 mg of N L(-1) d(-1) was achieved after 100 d treating a synthetic NH4+-rich wastewater. By gradually imposing higher loads on the reactor, the N elimination could be increased to about 1.8 g of N L(-1) d(-1) at 250 d. The resulting microbial community was compared with that of the inocula using general bacterial and AAOB- and planctomycete-specific PCR primers. Subsequently, the RBC reactor was shown to treat a sludge digestor effluent under suboptimal and strongly varying conditions. The RBC biocatalyst was also submitted to complete absence of oxygen in a fixed-film bioreactor (FFBR) and proved able to remove NH4+ with NO2- as electron acceptor (maximal 434 mg of NH4+-N (g of VSS)(-1) d(-1) on day 136). DGGE and real-time PCR analysis demonstrated that the RBC biofilm was dominated by members of the genus Nitrosomonas and close relatives of Kuenenia stuttgartiensis, a known AnAOB. The latter was enriched during FFBR operation, but AAOB were still present and the ratio planctomycetes/AAOB rRNA gene copies was about 4.3 after 136 d of reactor operation. Whether this relates to an active role of AAOB in the anoxic N removal process remains to be solved.

Bacteria, Aerobic↗

PubMed2005

Heme axial methionine fluxion in Pseudomonas aeruginosa Asn64Gln cytochrome c551.

Heme axial methionine ligands in ferricytochromes c552 from Hydrogenobacter thermophilus (HT) and Nitrosomonas europaea, both members of the cyt c8 family, display fluxional behavior. The ligand motion, proposed to be inversion at sulfur, results in an unusually small range of hyperfine shifts for heme substituents in these proteins. Herein, heme axial Met fluxion is induced in a structurally homologous cytochrome c551 from Pseudomonas aeruginosa (PA) by substituting heme pocket residue Asn64 with Gln. The mutant, PA-N64Q, displays a highly compressed range of heme substituent hyperfine shifts, temperature-dependent heme methyl resonance line broadening, low rhombic magnetic anisotropy, and a magnetic axes orientation consistent with Met orientational averaging. Analysis of NMR properties of PA-N64Q demonstrates that the heme pocket of the mutant resembles that of HT. This result confirms the importance of peripheral interactions and, in particular, residue 64 in determining axial Met orientation and heme electronic structure in proteins in the cyt c8 family.

Bacterial Proteins↗

PubMed2000

Phylogenetic analysis of rhizosphere-associated beta-subclass proteobacterial ammonia oxidizers in a municipal wastewater treatment plant based on rhizoremediation technology.

In wastewater treatment plants based on the rhizosphere zone (rhizoremediation technology), ammonia-oxidizing bacteria (AOB) play an important role in the removal of fixed nitrogen. However, the diversity of these bacteria in rhizoremediation wastewater treatment plants is largely unknown. We employed direct PCR amplification and cloning of 16S rRNA genes to determine the phylogenetic affiliation of AOB occurring in root and soil samples of a wastewater treatment plant (Merzdorf plant, Brandenburg, Germany). 16S rDNA clone libraries were screened by hybridization using an oligonucleotide probe specific for AOB of the beta subclass of proteobacteria. Comparative sequence analysis of all hybridization-positive clones revealed that the majority of rDNA sequences was affiliated to members of the genus Nitrosospira and formed a novel subcluster (SM cluster), whereas only three sequences were most closely related to Nitrosomonas species. Affiliation of the novel Nitrosospira-like sequences with those of isolates from soil and rhizosphere suggests that phylogenetic clusters reflect physiological differences between members of this genus.

Ammonia↗

PubMed2002

Use of natural zeolite to enhance nitrification in biofilter.

To enhance nitrification, natural zeolite and activated carbon were tested as a media in a biofilter to treat wastewater containing relatively high concentrations of TKN. Using those media, the adsorption isotherms for ammonium ion were compared; the K values for natural zeolite and activated carbon were found to be 0.5117 and 0.0006, respectively. In comparison of the performance of the two media, two identical, lab-scale biofilters were then operated for 4 months. The effect of NH3-N loading rates on the performance was investigated. The results showed that higher NH3-N removal efficiency and faster nitrification were achieved in the biofilter with natural zeolite throughout the experimental period. Nitrosomonas and Nitrobacter, two principal nitrifiers, in biofilm grown on two different media were counted and compared. Nitrobacter which is the more fragile of the two principal nitrifiers was outgrown in the biofilm on natural zeolite media. The reason for this may be due to the ammonium ion exchanging capacity of natural zeolite which provided the favorable environment for Nitrobacter.

Biodegradation, Environmental↗

PubMed2003

Molecular analysis of microbial communities in nitrification and denitrification reactors treating high ammonia leachate.

Molecular analysis of microbial populations in two bench-scale nitrification and denitrification reactors fed with high ammonia landfill leachate was conducted in this study by using DGGE, cloning, and FISH techniques in addition to classical efficiency control parameters. Nitrification tank was operated with a computer-controlled alkalinity dosing system to supply the alkalinity intermittently as consumed on the basis of on-line pH monitoring. By keeping the pH at 7.0 with this system, 99% nitrification efficiency and rates of about 0.14-0.18 mgNH4+-N/mgVSSday were obtained. Meanwhile, as ammonia oxidizing bacteria Nitrosomonas and Nitrosococcus mobilis-like cells and as nitrite oxidizing bacteria Nitrobacter-related cells were intensively indicated. Moreover, some aerobic denitrifiers as Thauera species were also identified. After the termination of pH adjustment in the preceding anaerobic reactors, nitrification tank was loaded with more biodegradable COD as a result of reduced COD removal in anaerobic reactors. Microbial diversity was immediately affected from this alteration and heterotrophic carbonaceous bacteria and aerobic denitrifiers have dominated. To provide the former high efficiencies, retention time has increased from 24 to 48 h and a second pump dosing HCl was included to the automatic control system. Subsequent to these precautions, numbers of ammonia (Nso190) and nitrite oxidizing bacteria (NIT3) were comparatively increased. In denitrification system, about 98% denitrification efficiencies were obtained at 2000 mg/L NOx-N concentrations if sodium acetate was supplied as carbon source. Meanwhile, with 20 gVSS/l biomass concentration, denitrification rates of about 1.34 mgNOx-N/mgVSSday were obtained. All sludge samples have represented similar DGGE patterns and Paraccoccus-related species were identified as dominant denitrifying bacteria.

Ammonia↗

PubMed2005

Comparative analysis of nitrifying bacteria in full-scale oxidation ditch and aerated nitrification biofilter by using fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE).

In this study, nitrification performances and composition of nitrifying populations in a full-scale oxidation ditch and a high-rate submerged media nitrification biofilter were comparatively analyzed. In addition to different reactor configurations, effects of differing operational conditions on the nitrification efficiency and bacterial diversity were also explored and evaluated thoroughly. In microbial analysis of sludge samples fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) techniques were used complementary to each other. The extended aeration oxidation ditch subjected to the study is operated as a nitrogen and phosphorus removal system consisting of anaerobic, anoxic, and aerobic zones. The high-rate submerged media aerated filter is operated as nitrification step following the conventional activated sludge unit and the nitrified wastewater is discharged to the sea without complete nitrogen removal. In situ hybridization results have indicated that Nitrosomonas-like ammonia oxidizing and Nitrospira-related nitrite oxidizing bacteria were intensively present in vigorous flocs in nitrification biofilter while carbonaceous bacteria belong to beta subclass of Proteobacteria were considerably dominant in oxidation ditch. Low quantities of nitrifiers in oxidation ditch were also confirmed by the dissimilarity in intensive bands between two systems obtained with DGGE analysis.

Aerobiosis↗

PubMed2026

Hypoosmolarity inhibits ammonia oxidation by terrestrial and freshwater Nitrosopumilaceae members.

Salinity strongly influences the physiology and distribution of nitrifying microorganisms, yet the effects of low salinity remain understudied. This study investigates the impact of hypoosmolarity on different groups of ammonia oxidizers in soil and freshwater reservoirs, as well as in pure culture isolates. In soil microcosms amended with ammonium, at low salinity levels (~120 μS/cm), comparable to values commonly found in pristine terrestrial and freshwater environments, the abundance of ammonia-oxidizing bacteria (AOB), dominated by Nitrosomonas oligotropha, significantly increased. In contrast, the growth of ammonia-oxidizing archaea (AOA), dominated by "Candidatus Nitrosotenuis" of the Nitrosopumilaceae family, was stimulated by high salinity (~760 μS/cm). In ammonium-fed freshwater microcosms, the abundance of AOB, dominated by N. oligotropha, significantly increased under both low (~170 μS/cm) and high salinity (~850 μS/cm) conditions. In the presence of allylthiourea (50 μM), used to inhibit bacterial ammonia oxidation, AOA were sensitive to low salinity in both soil and freshwater microcosms. Consistently, culture-dependent studies revealed marked growth inhibition of terrestrial AOA, especially members of Nitrosopumilaceae, under hypoosmolarity, unlike AOB and complete ammonia oxidizer (comammox) strains. Genomic analyses, along with transcriptomic studies, suggested that the sensitivity of AOA to hypoosmolarity stress was possibly due to a lack of osmoregulatory transport systems and their S-layer cell wall structure. Overall, this study indicates hypoosmolarity as an important factor shaping the ecological niches and distribution of ammonia oxidizers, as well as nitrification activities, in terrestrial and freshwater environments that are increasingly affected by intensified water cycles due to global change.

Ammonia↗

PubMed2005

Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes.

Prokaryotic chromosomes code for toxin-antitoxin (TA) loci, often in multiple copies. In E.coli, experimental evidence indicates that TA loci are stress-response elements that help cells survive unfavorable growth conditions. The first gene in a TA operon codes for an antitoxin that combines with and neutralizes a regulatory 'toxin', encoded by the second gene. RelE and MazF toxins are regulators of translation that cleave mRNA and function, in interplay with tmRNA, in quality control of gene expression. Here, we present the results from an exhaustive search for TA loci in 126 completely sequenced prokaryotic genomes (16 archaea and 110 bacteria). We identified 671 TA loci belonging to the seven known TA gene families. Surprisingly, obligate intracellular organisms were devoid of TA loci, whereas free-living slowly growing prokaryotes had particularly many (38 in Mycobacterium tuberculosis and 43 in Nitrosomonas europaea). In many cases, TA loci were clustered and closely linked to mobile genetic elements. In the most extreme of these cases, all 13 TA loci of Vibrio cholerae were bona fide integron elements located in the V.cholerae mega-integron. These observations strongly suggest that TA loci are mobile cassettes that move frequently within and between chromosomes and also lend support to the hypothesis that TA loci function as stress-response elements beneficial to free-living prokaryotes.

Bacterial Toxins↗

PubMed1977

Purification and some properties of cytochrome c-552 from an extreme thermophile, Thermus thermophilus HB8.

A c-type cytochrome, cytochrome c-552, from a soluble fraction of an extreme thermophile, Thermus thermophilus HB8, was highly purified and its properties investigated. The absorption peaks were at 552, 522, and 417 nm in the reduced form, and at 408 nm in the oxidized form. The isoelectric point was at PH 10.8, the midpoint redox potential was about +0.23 V, and the molecular weight was about 15,000. The cytochrome c-552 was highly thermoresistant. The cytochrome reacted rapidly with pseudomonas aeruginosa nitrite reductase [EC 1.9.3.2], but slowly with bovine cytochrome oxidase [EC 1.9.3.1], yeast cytochrome c peroxidase [EC 1.11.1.5], or Nitrosomonas europaea hydroxylamine-cytochrome c reductase [EC 1.7.3.4].

Cytochrome c Group↗

PubMed1999

RFLP of rRNA genes and sequencing of the 16S-23S rDNA intergenic spacer region of ammonia-oxidizing bacteria: a phylogenetic approach.

It has been established that 16S rRNA gene-based phylogeny gives a low resolution between members of the chemoautotrophic ammonia-oxidizing bacteria (AOB) belonging to the beta-subclass of the Proteobacteria. In this study, 12 isolates of AOB were ribotyped, and the sequences of the 16S-23S rDNA intergenic spacer region (ISR) were determined and used in a phylogenetic study. 16S and 23S rDNA ribotyping revealed that the AOB studied contain only one rrn operon per genome, in contrast to most bacteria, which have 5-10 copies of the rRNA genes per genome. It is likely that the presence of only one set of rRNA genes is related to the slow growth of the AOB. The 16S and 23S rRNA genes of the AOB were shown to be arranged in the classical way: a 16S rRNA gene, an ISR and a 23S rRNA gene. Despite the close phylogenetic relationship among the AOB, the relative location of the rRNA genes in the genome appears to vary considerably. The size of the ISR was approximately 400 bp in the Nitrosomonas isolates and 645-694 bp in the Nitrosospira isolates, suggesting a species-specific size difference in the ISR. The ISR contained two potential tRNA genes in the 5' end in all isolates studied. The similarity values between the ISR sequences of the AOB are low (42.9-96.2%) compared with the 16S rDNA sequence similarity values, and therefore the ISR sequences are valuable as a complementary phylogenetic tool in combination with 16S rRNA gene sequences. The phylogenetic analysis of the AOB based on ISR sequences confirms the 16S rRNA gene-based phylogeny but has the benefit of giving a higher resolution.

Ammonia↗

PubMed1993

The phylogeny of autotrophic ammonia-oxidizing bacteria as determined by analysis of 16S ribosomal RNA gene sequences.

Partial sequences of the 16S ribosomal RNA genes of eleven autotrophic ammonia-oxidizing bacteria were determined by PCR amplification from small amounts of heat-lysed biomass followed by direct sequencing of PCR products. The sequences were aligned with those of representative Proteobacteria and phylogenetic trees inferred using both parsimony and distance matrix methods. This confirmed that the autotrophic ammonia-oxidizers comprise two major lines of descent within the Proteobacteria. Nitrosomonas spp., Nitrosococcus mobilis, and strains of Nitrosovibrio, Nitrosospira and Nitrosolobus were located in the beta-subdivision. The recovery of Nitrosococcus oceanus strains as a deep branch in the gamma-subdivision supported the RNA catalogue data which had indicated that the genus Nitrosococcus is polyphyletic. The autotrophic ammonia-oxidizing bacteria of the beta-Proteobacteria formed a coherent group which is interpreted as representing a single family. Within this clade, the genera Nitrosovibrio, Nitrosospira and Nitrosolobus exhibited very high levels of homology in their 16S ribosomal RNA gene sequences and can be accommodated within a single genus. Separation of these genera is currently based entirely on gross morphological differences and these can now be considered more appropriate for the identification of species within this group. It is therefore proposed that Nitrosolobus, Nitrosovibrio and Nitrosospira strains be reclassified in a single genus for which the name Nitrosospira has priority.

Ammonia↗

PubMed1997

Heterologous expression of heterotrophic nitrification genes.

Paracoccus denitrificans is a heterotrophic organism capable of oxidizing ammonia to nitrite during growth on an organic carbon and energy source. This pathway, termed heterotrophic nitrification, requires the concerted action of an ammonia monooxygenase (AMO) and hydroxylamine oxidase (HAO). The genes required for heterotrophic nitrification have been isolated by introducing a Pa. denitrificans genomic library into Pseudomonas putida and screening for the accumulation of nitrite. In contrast to the situation in chemolithoautotrophic ammonia oxidizers, the genes encoding AMO and HAO are present in single linked copies in the genome of Pa. denitrificans. AMO from Pa. denitrificans expressed in Ps. putida is capable of oxidizing ethene (ethylene) to epoxyethane (ethylene oxide), which is indicative of a relaxed substrate specificity. Further, when expressed in the methylotroph Methylobacterium extorquens AM1, the AMO endows on this organism the ability to grow on ethene and methane. Thus, the Pa. denitrificans AMO is capable of oxidizing methane to methanol, as is the case for the AMO from Nitrosomonas europaea. The heterotrophic nitrification genes are moderately toxic in M. extorquens, more toxic in Ps. putida, and non-toxic in Escherichia coli. Toxicity is due to the activity of the gene products in M. extorquens, and both expression and activity in Ps. putida. This is the first time that the genes encoding an active AMO have been expressed in a heterologous host.

Ammonia↗

PubMed2002

Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing.

Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3.5) microcosms that were exposed to (13)CH(3)OH or (13)CH(4). Distinct (13)C-labelled DNA ((13)C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the (13)C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the (13)C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH(3)OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the beta-subclass of the PROTEOBACTERIA: Analysis of AOB-selective 16S rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the (13)C-DNA fractions, suggesting certain AOB assimilated a significant proportion of (13)CO(2), possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the (13)C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the beta-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the (13)CH(3)OH and (13)CH(4) SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ.

Ammonia↗

PubMed2003

16S rRNA and amoA-based phylogeny of 12 novel betaproteobacterial ammonia-oxidizing isolates: extension of the dataset and proposal of a new lineage within the nitrosomonads.

The phylogenetic relationship of 12 ammonia-oxidizing isolates (eight nitrosospiras and four nitrosomonads), for which no gene sequence information was available previously, was investigated based on their genes encoding 16S rRNA and the active site subunit of ammonia monooxygenase (AmoA). Almost full-length 16S rRNA gene sequences were determined for the 12 isolates. In addition, 16S rRNA gene sequences of 15 ammonia-oxidizing bacteria (AOB) published previously were completed to allow for a more reliable phylogeny inference of members of this guild. Moreover, sequences of 453 bp fragments of the amoA gene were determined from 15 AOB, including the 12 isolates, and completed for 10 additional AOB. 16S rRNA gene and amoA-based analyses, including all available sequences of AOB pure cultures, were performed to determine the position of the newly retrieved sequences within the established phylogenetic framework. The resulting 16S rRNA gene and amoA tree topologies were similar but not identical and demonstrated a superior resolution of 16S rRNA versus amoA analysis. While 11 of the 12 isolates could be assigned to different phylogenetic groups recognized within the betaproteobacterial AOB, the estuarine isolate Nitrosomonas sp. Nm143 formed a separate lineage together with three other marine isolates whose 16S rRNA sequences have not been published but have been deposited in public databases. In addition, 17 environmentally retrieved 16S rRNA gene sequences not assigned previously and all originating exclusively from marine or estuarine sites clearly belong to this lineage.

Ammonia↗

PubMed2002

Evolution of sucrose synthesis.

Cyanobacteria and proteobacteria (purple bacteria) are the only prokaryotes known to synthesize sucrose (Suc). Suc-P synthase, Suc-phosphatase (SPP), and Suc synthase activities have previously been detected in several cyanobacteria, and genes coding for Suc-P synthase (sps) and Suc synthase (sus) have been cloned from Synechocystis sp. PCC 6803 and Anabaena (Nostoc) spp., respectively. An open reading frame in the Synechocystis genome encodes a predicted 27-kD polypeptide that shows homology to the maize (Zea mays) SPP. Heterologous expression of this putative spp gene in Escherichia coli, reported here, confirmed that this open reading frame encodes a functional SPP enzyme. The Synechocystis SPP is highly specific for Suc-6(F)-P (K(m) = 7.5 microM) and is Mg(2+) dependent (K(a) = 70 microM), with a specific activity of 46 micromol min(-1) mg(-1) protein. Like the maize SPP, the Synechocystis SPP belongs to the haloacid dehalogenase superfamily of phosphatases/hydrolases. Searches of sequenced microbial genomes revealed homologs of the Synechocystis sps gene in several other cyanobacteria (Nostoc punctiforme, Prochlorococcus marinus strains MED4 and MIT9313, and Synechococcus sp. WH8012), and in three proteobacteria (Acidithiobacillus ferrooxidans, Magnetococcus sp. MC1, and Nitrosomonas europaea). Homologs of the Synechocystis spp gene were found in Magnetococcus sp. MC1 and N. punctiforme, and of the Anabaena sus gene in N. punctiforme and N. europaea. From analysis of these sequences, it is suggested that Suc synthesis originated in the proteobacteria or a common ancestor of the proteobacteria and cyanobacteria.

Archaea↗