Phylogeny of anoxygenic filamentous phototrophic bacteria of the family Oscillochloridaceae as inferred from comparative analyses of the rrs, cbbL, and nifH genes

Phylogeny of anoxygenic filamentous phototrophic bacteria (AFPB) of the family Oscillochloridaceae (Oscillochloris trichoides DG6^T and the recently isolated strains Oscillochloris sp. R and C6) was studied based on comparative analyses of the genes coding for 16S rRNA (rrs), ribulose-1,5-bisphosphate carboxylase/oxygenase (cbbL), and nitrogenase (nifH). The sequences of the genes studied proved to be identical in the three strains, which is in agreement with data obtained earlier that showed a lack of differentiating phenotypic distinctions between these strains; therefore, it is proposed that the new strains should be identified as representatives of the species O. trichoides. Using an earlier designed system of oligonucleotide primers and a specially designed additional primer, fragments of the cbbL genes of the “red-like” form I RuBisCO were amplified and sequenced for all of the O. trichoides strains. Analysis of the cbbL genes suggested a separate position of the bacteria studied in the phylogenetic tree, where O. trichoides strains formed an independent branch, which, apart from this species, also included the only studied species of gram-positive facultatively chemoautotrophic bacteria, Sulfobacillus acidophilus. In the phylogenetic tree inferred from the analysis of nifH genes, the bacteria under study also formed a new separate branch, deviating near the root, which indicated a lack of relatedness between them and other phototrophic bacteria. The data obtained support the conclusion that AFPB has an ancient origin and their allocation as one of the main evolutionary lineages of eubacteria, which was made based on the analysis of ribosomal genes.     

Draft genome sequence of the anoxygenic filamentous phototrophic bacterium Oscillochloris trichoides subsp. DG-6

Oscillochloris trichoides is a mesophilic, filamentous, photoautotrophic, nonsulfur, diazotrophic bacterium which is capable of carbon dioxide fixation via the reductive pentose phosphate cycle and possesses no assimilative sulfate reduction. Here, we present the draft genome sequence of Oscillochloris trichoides subsp. DG-6, the type strain of the species, which has permitted the prediction of genes for carbon and nitrogen metabolism and for the light-harvesting apparatus.     

Characterization of amplified polymerase chain reaction glnB and nifH gene fragments of nitrogen-fixing Burkholderia species

AIMS: To clone and sequence polymerase chain reaction (PCR)-amplified glnB and nifH genes of the nitrogen-fixing bacteria Burkholderia brasilensis strain M130, B. tropicalis strain PPe8 and B. kururiensis strain KP23. METHODS AND RESULTS: The glnB and nifH gene fragments were amplified by PCR using universal degenerated primers. A very high percentage of similarity for the nifH (100%) and glnB (96%) genes was observed between strains M130 and KP23. A similarity of 100% for the nifH gene was also observed between strains M130 and PPe8. However, the identity for the glnB gene was 98% and the similarity 88%. The phylogenetic tree of the nifH gene showed a very high degree of similarity to the 16S rDNA gene. CONCLUSIONS: The nitrogen-fixing bacteria of the Burkholderia genus formed a cluster separated from the other species of the genus mainly when the nifH rather than the glnB gene was used to construct the phylogenetic tree. Significance and Impact of the Study: Knowledge of the nifH and glnB gene sequences of B. brasilensis, B. tropicalis and B. kururiensis will support new studies on the diversity of these diazotrophs in natural environments.     

A study of the content of pigments in the light-harvesting antenna of the green bacterium from the new family Oscillochloridaceae

The properties of the light-harvesting superantenna of the photosynthesizing bacteria from the new family of green filamentous bacteria Oscillochloridaceae were investigated by optical spectroscopy. The antenna of Oscillochloris trichoides consists of peripheral chlorosomal and membrane subantennas. A method of isolation of Osc. trichoides chlorosomal antenna was developed using the chaothropic agent sodium thiocyanate, which simultaneously acts to stabilize chlorosomal activity. An analysis of the second derivatives of the absorption spectra of isolated chlorosomes and their acetone-methanol extracts suggested that BChl c was a predominant light-harvesting pigment in Osc. trichoides chlorosomes. Besides, it was found that, in addition to the BChl c-antenna, chlorosomes contain a minor BChl a-antenna. It was shown that the membrane BChl a-subantenna is a light-harvesting complex with absorption maxima in the near infrared region at 805 and 860 nm. Analysis of the spectral data obtained suggested that the Osc. trichoides chlorosomal antenna resembles those from Chlorobiaceae species, whereas the membrane B805-860 BChl a antenna of Osc. trichoides is close to the membrane B808-866 BChl a antenna of Chloroflexaceae species.     

"Green-like" and "red-like" RubisCO cbbL genes in Rhodobacter azotoformans

We found that Rhodobacter azotoformans IFO 16436T contains two different cbbL genes coding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunits. One gene is located within a "green-like" group of the RubisCO phylogenetic tree, and the other is located within a "red-like" group. This is the first report that one organism contains both green-like and red-like RubisCO genes. Moreover, by PCR using primers which amplify two green-like and red-like cbbL genes alternatively and dot blot hybridization, we demonstrated that Rhodobacter blasticus, Rhodobacter capsulatus, and Rhodobacter veldkampii possess only green-like cbbL genes, and Rhodobacter sphaeroides possesses only a red-like cbbL gene. In the cbbL phylogenic analysis, R. spaeroides and R. azotoformans 1 (red-like) formed a cluster within the red-like group, and R. capsulatus, R. azotoformans 2 (green-like), R. blasticus, and R. veldkampii formed a cluster within the green-like group. This suggests that red-like cbbL genes of Rhodobacter species were derived from one ancestor, and green-like cbbL genes were derived from another ancestor. On the other hand, molecular phylogeny of the bacteria indicates that R. veldkampii, which has only a green-like cbbL gene, is the earliest evolved Rhodobacter species and that R. azotoformans and R. sphaeroides, which have red-like cbbL genes, are the latest evolved. Consequently, the following hypothesis is proposed: the common ancestor of Rhodobacter had a green-like cbbL gene, the common ancestor of R. azotoformans and R. sphaeroides subsequently obtained a red-like cbbL gene by a horizontal gene transfer, and the ancestor of R. sphaeroides later lost the green-like cbbL gene.     

A comparative study of the fluorescence properties of the chlorosomal antenna of the green bacterium from the family Oscillochloridaceae and the members from two other families of green bacteria

The fluorescence properties of bacteriochlorophylls (BChl) of the chlorosomal light-harvesting antenna of Oscillochloris trichoides (strain DG-6) from a new family of green filamentous bacteria Oscillochloridaceae were investigated in comparison with green bacteria from two other families. A strong dependence of the fluorescence intensity of chlorosomal bacteriochlorophyll c of Osc. trichoides on the redox potential of medium was found, which previously was observed only in green sulfur bacteria. The presence of BChl a in chlorosomes did not appear in their absorption spectra but was visualized by fluorescence spectroscopy at 77 K. From the comparative analysis of fluorescence spectral data for the chlorosomal light-harvesting antenna of Osc. trichoides and similar spectral data for green bacteria from two other families, it was concluded that, in some fluorescence spectral features (spectral position of bacteriochlorophyll c/a fluorescence bands; shape and full width at half maximum fluorescence band of chlorosomal bacteriochlorophyll c; the Stokes shift value of bacteriochlorophyll c band; a high molar ratio of bacteriochlorophyll c : bacteriochlorophyll a in chlorosomes that makes the bacteriochlorophyll a fluorescence band unresolved at room temperature; and highly redox-dependent fluorescence intensity of chlorosomal bacteriochlorophyll c), Osc. trichoides chlorosomes are close to the chlorosomal antenna of Chlorobiaceae species.     

Diversity of green-like and red-like ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes (cbbL) in differently managed agricultural soils

A PCR-based approach was developed to detect ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) form I large-subunit genes (cbbL) as a functional marker of autotrophic bacteria that fix carbon dioxide via the Calvin-Benson-Bassham cycle. We constructed two different primer sets, targeting the green-like and red-like phylogenetic groups of cbbL genes. The diversity of these cbbL genes was analyzed by the use of three differently managed agricultural soils from a long-term field experiment. cbbL gene fragments were amplified from extracted soil DNAs, and PCR products were cloned and screened by restriction fragment length polymorphism analysis. Selected unique cbbL clones were sequenced and analyzed phylogenetically. The green-like cbbL sequences revealed a very low level of diversity, being closely related to the cbbL genes of Nitrobacter winogradskyi and Nitrobacter vulgaris. In contrast, the red-like cbbL gene libraries revealed a high level of diversity in the two fertilized soils and less diversity in unfertilized soil. The majority of environmental red-like cbbL genes were only distantly related to already known cbbL sequences and even formed separate clusters. In order to extend the database of available red-like cbbL sequences, we amplified cbbL sequences from bacterial type culture strains and from bacterial isolates obtained from the investigated soils. Bacterial isolates harboring the cbbL gene were analyzed phylogenetically on the basis of their 16S rRNA gene sequences. These analyses revealed that bacterial genera such as Bacillus, Streptomyces, and Arthrobacter harbor red-like cbbL genes which fall into the cbbL gene clusters retrieved from the investigated soils.     

Carbon metabolism of filamentous anoxygenic phototrophic bacteria of the family Oscillochloridaceae

The carbon metabolism of representatives of the family Oscillochloridaceae (Oscillochloris trichoides DG6 and the recent isolates Oscillochloris sp. R, KR, and BM) has been studied. Based on data from an inhibitory analysis of autotrophic CO2 assimilation and measurements of the activities of the enzymes involved in this process, it is concluded that, in all Oscillochloris strains, CO2 fixation occurs via the operation of the Calvin cycle. Phosphoenolpyruvate (PEP), which is formed in this cycle, can be involved in the metabolism via the following reaction sequence: PEP (+ CO2) --> oxalacetate --> malate --> fumarate --> succinate --> succinyl-CoA (+ CO2) --> 2-oxoglutarate (+ CO2) --> isocitrate. Acetate, utilized as and additional carbon source, can be carboxylated to pyruvate by pyruvate synthase and further involved in the metabolism via the above reaction sequence. Propionyl-CoA synthase and malonyl-CoA reductase, the key enzymes of the 3-hydroxypropionate cycle, have not been detected in Oscillochloris representatives.     

Molecular diversity of nitrogen-fixing bacteria from the Tibetan Plateau, China

To understand the composition and structure of nitrogen-fixing bacterial communities from the Sanjiangyuan Nature Reserve on the Tibetan Plateau, the molecular diversity of nifH genes from soil obtained at six sites was examined using a PCR-based cloning approach. Six samples were collected from different regions at an altitude of 3907-4824 m above sea level, and a principal component analysis (PCA) showed that they had different biogeochemical properties. A total of 446 clones and 162 unique RFLP patterns were found. PCA of the RFLP patterns and their biogeochemical parameters showed that the content of soil organic carbon (C), total nitrogen (N) and altitude were the most important factors affecting the nitrogen-fixing bacteria community. Fifty-nine nifH clones were sequenced and their nucleotide identity varied from 64% to 98%, subdivisible into four groups in our phylogenetic tree. Some of the clone sequences were related to nifH genes belonging to four phylogenetic subdivisions (alpha, beta, gamma and delta subclasses of the Proteobacteria), while most of the clones were closely related to the genes of the uncultured bacteria. The tree also showed that the sequence distributions were not clearly related to the sample sites.     

Chlorobaculum macestae sp. nov., a new green sulfur bacterium

The investigated green sulfur bacterium, strain M, was isolated from a sulfidic spring on the Black Sea Coast of the Caucasus. The cells of strain M are straight or curved rods 0.6-0.9 x 1.8-4.2 microm in size. According to the cell wall structure, the bacteria are gram-negative. Chlorosomes are located along the cell periphery. Strain M is an obligate anaerobe capable of photoautotrophic growth on sulfide, thiosulfate, and H2. It utilizes ammonium, urea, casein hydrolysate, and N2 as nitrogen sources and sulfide, thiosulfate, and elemental sulfur as sulfur sources. Bacteriochlorophyll c and the carotenoid chlorobactene are the main pigments. The optimal growth temperature is 25-28 degrees C; the optimal pH is 6.8. The strain does not require NaCl. Vitamin B12 stimulates growth. The content of the G+C base pairs in the DNA of strain M is 58.3 mol %. In the phylogenetic tree constructed on the basis of analysis of nucleotide sequences of 16S rRNA genes, strain M forms a separate branch, which occupies an intermediate position between the phylogenetic cluster containing representatives of the genus Chlorobaculum (94.9-96.8%) and the cluster containing species of the genus Chlorobium (94.1-96.5%). According to the results of analysis of the amino acid sequence corresponding to the fmo gene, strain M represents a branch which, unlike that in the "ribosomal" tree, falls into the cluster of the genus Chlorobaculum (95.8-97.2%). Phylogenetic analysis of the amino acid sequence corresponding to the nifH gene placed species of the genera Chlorobaculum and Chlorobium into a single cluster, whereas strain M formed a separate branch. The results obtained allow us to describe strain M as a new species of the genus Chlorobaculum. Chlorobaculum macestae sp. nov.     

Phylogenetically diverse groups of Bradyrhizobium isolated from nodules of Crotalaria spp., Indigofera spp., Erythrina brucei and Glycine max growing in Ethiopia

Ethiopian Bradyrhizobium strains isolated from root nodules of Crotalaria spp., Indigofera spp., Erythina brucei and soybean (Glycine max) represented genetically diverse phylogenetic groups of the genus Bradyrhizobium. Strains were characterized using the amplified fragment length polymorphism fingerprinting technique (AFLP) and multilocus sequence analysis (MLSA) of core and symbiotic genes. Based on phylogenetic analyses of concatenated recA-glnII-rpoB-16S rRNA genes sequences, Bradyrhizobium strains were distributed into fifteen phylogenetic groups under B. japonicum and B. elkanii super clades. Some of the isolates belonged to the species B. yuanmingense, B. elkanii and B. japonicum type I. However, the majority of the isolates represented unnamed Bradyrhizobium genospecies and of these, two unique lineages that most likely represent novel Bradyrhizobium species were identified among Ethiopian strains. The nodulation nodA gene sequence analysis revealed that all Ethiopian Bradyrhizobium isolates belonged to nodA sub-clade III.3. Strains were further classified into 14 groups together with strains from Africa, as well as some originating from the other tropical and subtropics regions. Strains were also clustered into 14 groups in nodY/K phylogeny similarly to the nodA tree. The nifH phylogenies of the Ethiopian Bradyrhizobium were generally also congruent with the nodA gene phylogeny, supporting the monophyletic origin of the symbiotic genes in Bradyrhizobium. The phylogenies of nodA and nifH genes were also partially congruent with that inferred from the concatenated core genes sequences, reflecting that the strains obtained their symbiotic genes vertically from their ancestor as well as horizontally from more distantly related Bradyrhizobium species.     

Molecular evidence that the capacity for endosporulation is universal among phototrophic heliobacteria

Although enrichment cultures for anoxygenic phototrophic heliobacteria commonly contain sporulating cells, once strains of heliobacteria are obtained in pure culture, they all but cease to sporulate. In fact, some species of heliobacteria have never been observed to sporulate. Thus, despite their phylogenetic connection to endospore-forming bacteria, the question of sporulation capacity in heliobacteria remains open. We have investigated this problem using PCR and Southern hybridization as tools and show here that all recognized species of heliobacteria tested, as well as several unclassified strains, contain homologs to the ssp genes of Clostridium and Bacillus species, genes that encode key sporulation-specific proteins. It can therefore be concluded that as a group, heliobacteria are likely all to be endospore-forming bacteria in agreement with their phylogenetic placement within the 'low GC' Gram-positive bacteria.     

Nodulation of Lupinus albus by strains of Ochrobactrum lupini sp. nov

The nodulation of legumes has for more than a century been considered an exclusive capacity of a group of microorganisms commonly known as rhizobia and belonging to the alpha-Proteobacteria. However, in the last 3 years four nonrhizobial species, belonging to alpha and beta subclasses of the Proteobacteria, have been described as legume-nodulating bacteria. In the present study, two fast-growing strains, LUP21 and LUP23, were isolated from nodules of Lupinus honoratus. The phylogenetic analysis based on the 16S and 23S rRNA gene sequences showed that the isolates belong to the genus Ochrobactrum. The strains were able to reinfect Lupinus plants. A plasmid profile analysis showed the presence of three plasmids. The nodD and nifH genes were located on these plasmids, and their sequences were obtained. These sequences showed a close resemblance to the nodD and nifH genes of rhizobial species, suggesting that the nodD and nifH genes carried by strain LUP21T were acquired by horizontal gene transfer. A polyphasic study including phenotypic, chemotaxonomic, and molecular features of the strains isolated in this study showed that they belong to a new species of the genus Ochrobactrum for which we propose the name Ochrobactrum lupini sp. nov. Strain LUP21T (LMG 20667T) is the type strain.     

Klebsiella variicola, a novel species with clinical and plant-associated isolates

A new Klebsiella species, K. variicola, is proposed on the basis of total DNA-DNA hybridization, on the monophyly observed in the phylogenetic analysis derived from the sequences of rpoB, gyrA, mdh, infB, phoE and nifH genes and on distinct phenotypic traits. The bacteria from this new species seem to be genetically isolated from K. pneumoniae strains, do not ferment adonitol and were obtained from plants (such as banana, rice, sugar cane and maize) and hospitals. The type strain is F2R9T (= ATCC BAA-830T = CFNE 2004T).     

Novel facultative anaerobic acidotolerant Telmatospirillum siberiense gen. nov. sp. nov. isolated from mesotrophic fen

Three facultative anaerobic acidotolerant Gram-negative motile spirilla strains designated 26-4b1, 26-2 and K-1 were isolated from mesotrophic Siberian fen as a component of methanogenic consortia. The isolates were found to grow chemoorganotrophically on several organic acids and glucose under anoxic and low oxygen pressure in the dark, tolerant up to 5kPa of oxygen. At low oxygen supply, faint autotrophic growth on the H(2):CO(2) mixture was also observed. All three isolates were able to fix N(2). Major cellular fatty acids were 18:1 omega7c, 17:0 cyclopropane and 16:0. Phylogenetic analyses of the 16S rRNA gene sequences revealed that they formed a deep branch within the family Rhodospirillaceae of the Alphaproteobacteria with the highest similarity of 90.9-92.5% with members of genera Phaeospirillum and Magnetospirillum. Phylogenetic study of nifH (nitrogenase) and cbbL (RuBisCO) amino acid sequence identities confirmed that the new isolates represent a novel group. Based on the phylogenetic analyses and distinct phenotypic characteristics, we are of the opinion that strains 26-4b1, 26-2 and K-1 represent a new species of a novel genus for which the name Telmatospirillum siberiense gen. nov. sp. nov. is proposed.     

Factors controlling the biosynthesis of chlorosome antenna bacteriochlorophylls in green filamentous anoxygenic phototrophic bacteria of the family Oscillochloridaceae

We determined the concentrations of bacteriochlorophylls (BChl) in the light-harvesting antennae of Oscillochloris trichoides (of the family Oscillochloridaceae belonging to green filamentous mesophilic bacteria) cultivated either with gabaculine, an inhibitor of the C-5 pathway of BChl biosynthesis in a number of bacteria, or at various illumination intensities. We determined the BChl c: BChl a molar ratios in intact cells, in chlorosome-membrane complexes, and in isolated chlorosomes. We revealed that BChl c synthesis in Osc. trichoides was more gabaculine-sensitive than BChl a synthesis. Accordingly, an increase in gabaculine concentrations in the medium resulted in a decrease in the BChl c: BChl a ratio in the tested samples. We suggest that BChl synthesis in Osc. trichoides proceeds via the C-5 pathway, similar to representatives of other families of green bacteria (Chlorobium limicola and Chloroflexus aurantiacus). We demonstrated that the BChl c: BChl a ratio in the chlorosomes varied from 55 : 1 to 110 : 1, depending on light intensity. This ratio is, therefore, closer to that of Chlorobiaceae, and it significantly exceeds the BChl c: BChl a ratio in Chloroflexaceae.     

Study of nucleotide sequences of nifH genes in methanotrophic bacteria

Using a previously developed primer system, nifH gene fragments 450 nucleotides long were amplified, cloned, and sequenced for representatives of nitrogen-fixing methanotrophic bacteria of the genera Methylococcus, Methylocystis and Methylosinus. Fragments of nifH genes were also detected and sequenced in representatives of the genera Methylomonas and Methylobacter, which were not considered diazotrophs until recently. Phylogenetic analysis revealed remoteness of nifH genes sequences of methanotroph types I and II. At the same time, close relationship was found between nifH of type I methanotrophs and representatives of gamma-proteobacteria and between nifH genes of type II methanotrophs and representatives of alpha-proteobacteria. The results obtained in this study are in good accordance with the data of phylogenetic analysis based on 16S rRNA sequence comparison with the only exception of Methylococcus capsulatus strains, whose nifH genes proved to be closely related to nifH genes of Methylocystis and Methylosinus representatives. Our findings extend the database of primary sequences of nifH genes and allow the contribution of methanotrophs to the process of nitrogen fixation to be estimated.     

Diverse rhizobia that nodulate two species of Kummerowia in China

A total of 63 bacterial strains were isolated from root nodules of Kummerowia striata and K. stipulacea grown in different geographic regions of China. These bacteria could be divided into fast-growing (FG) rhizobia and slow-growing (SG) rhizobia according to their growth rate. Genetic diversity and taxonomic relationships among these rhizobia were revealed by PCR-based 16 S rDNA RFLP and sequencing, 16 S-IGS RFLP, SDS-PAGE of whole cell soluble proteins, BOX-PCR and symbiotic gene (nifH/nodC) analyses. The symbiotic FG strains were mainly isolated from temperate regions and they were identified as four genomic species in Rhizobium and Sinorhizobium meliloti based on the consensus of grouping results. The SG strains were classified as five genomic species within Bradyrhizobium and they were mainly isolated fron the subtropic and tropical regions. The phylogenetic analyses of nifH and nodC genes showed relationships similar to that of 16 S rDNA but the symbiotic genes of Bradyrhizobium strains isolated from Kummerowia were distinct from those isolated from Arachis and soybean. These results offered evidence for rhizobial biogeography and demonstrated that the Kummerowia-nodulating ability might have evolved independently in different regions in association with distinctive genomic species of rhizobia.     

Legume symbiotic nitrogen fixation by beta-proteobacteria is widespread in nature

Following the initial discovery of two legume-nodulating Burkholderia strains (L. Moulin, A. Munive, B. Dreyfus, and C. Boivin-Masson, Nature 411:948-950, 2001), we identified as nitrogen-fixing legume symbionts at least 50 different strains of Burkholderia caribensis and Ralstonia taiwanensis, all belonging to the beta-subclass of proteobacteria, thus extending the phylogenetic diversity of the rhizobia. R. taiwanensis was found to represent 93% of the Mimosa isolates in Taiwan, indicating that beta-proteobacteria can be the specific symbionts of a legume. The nod genes of rhizobial beta-proteobacteria (beta-rhizobia) are very similar to those of rhizobia from the alpha-subclass (alpha-rhizobia), strongly supporting the hypothesis of the unique origin of common nod genes. The beta-rhizobial nod genes are located on a 0.5-Mb plasmid, together with the nifH gene, in R. taiwanensis and Burkholderia phymatum. Phylogenetic analysis of available nodA gene sequences clustered beta-rhizobial sequences in two nodA lineages intertwined with alpha-rhizobial sequences. On the other hand, the beta-rhizobia were grouped with free-living nitrogen-fixing beta-proteobacteria on the basis of the nifH phylogenetic tree. These findings suggest that beta-rhizobia evolved from diazotrophs through multiple lateral nod gene transfers.     

Diversity and expression of cyanobacterial hupS genes in pure cultures and in a nitrogen-limited phototrophic biofilm

A PCR was developed for conserved regions within the cyanobacterial small subunit uptake hydrogenase (hupS) gene family. These primers were used to PCR amplify partial hupS sequences from 15 cyanobacterial strains. HupS clone libraries were constructed from PCR-amplified genomic DNA and reverse-transcribed mRNA extracted from phototrophic biofilms cultivated under nitrate-limiting conditions. Partial hupS gene sequences derived from cyanobacteria, some of which were not previously known to contain hup genes were used for phylogenetic analysis. Phylogenetic trees constructed with partial hupS genes were congruent with those based on 16S rRNA genes, indicating that hupS sequences can be used to identify cyanobacteria expressing hup. Sequences from heterocystous and nonheterocystous cyanobacteria formed two separate clusters. Analysis of clone library data showed a discrepancy between the presence and the activity of cyanobacterial hupS genes in phototrophic biofilms. The results showed that the hupS gene can be used to characterize the diversity of natural populations of diazotrophic cyanobacteria, and to characterize gene expression patterns of individual species and strains.