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ABSTRACT Prokaryotes contribute to the global sulfur cycle by using diverse sulfur compounds as sulfur sources or electron acceptors. In this study, we report that a nitrogenase-like enzyme (NFL) and a radical SAM enzyme (RSE) are involved in the novel anaerobic assimilation pathway of a sulfonate, isethionate, in the photosynthetic bacterium Rhodobacter capsulatus. The nflHDK genes for NFL are localized at a locus containing genes for known sulfonate metabolism in the genome. A gene nflB encoding an RSE is present just upstream of nflH, forming a small gene cluster nflBHDK. Mutants lacking any nflBHDK genes are incapable of growing with isethionate as the sole sulfur source under anaerobic photosynthetic conditions, indicating that all four NflBHDK proteins are essential for the isethionate assimilation pathway. Heterologous expression of the islAB genes encoding a known isethionate lyase that degrades isethionate to sulfite and acetaldehyde restored the isethionate-dependent growth of a mutant lacking nflDK, indicating that the enzyme encoding nflBHDK is involved in an isethionate assimilation reaction to release sulfite. Furthermore, the heterologous expression of nflBHDK and ssuCAB encoding an isethionate transporter in the closely related species R. sphaeroides, which does not have nflBHDK and cannot grow with isethionate as the sole sulfur source, conferred isethionate-dependent growth ability to this species. We propose to rename nflBHDK as isrBHDK (isethionate reductase). The isrBHDK genes are widely distributed among various prokaryote phyla. Discovery of the isethionate assimilation pathway by IsrBHDK provides a missing piece for the anaerobic sulfur cycle and for understanding the evolution of ancient sulfur metabolism.IMPORTANCENitrogenase is an important enzyme found in prokaryotes that reduces atmospheric nitrogen to ammonia and plays a fundamental role in the global nitrogen cycle. It has been noted that nitrogenase-like enzymes (NFLs), which share an evolutionary origin with nitrogenase, have evolved to catalyze diverse reactions such as chlorophyll biosynthesis (photosynthesis), coenzyme F430 biosynthesis (methanogenesis), and methionine biosynthesis. In this study, we discovered that an NFL with unknown function in the photosynthetic bacterium Rhodobacter capsulatus is a novel isethionate reductase (Isr), which catalyzes the assimilatory degradation of isethionate, a sulfonate, releasing sulfite used as the sulfur source under anaerobic conditions. Isr is widely distributed among various bacterial phyla, including intestinal bacteria, and is presumed to play an important role in sulfur metabolism in anaerobic environments such as animal guts and microbial mats. This finding provides a clue for understanding ancient metabolism that evolved under anaerobic environments at the dawn of life.

Non-cyanobacterial diazotrophs (NCDs) are widespread and active in marine waters. The carbon and low-oxygen (O2) conditions required for their N2 fixation may be encountered on marine particles, while a putative role of light remains uninvestigated. This study explored factors that regulate N2 fixation in Rhodopseudomonas sp. BAL398--a anoxygenic phototrophic bacterium isolated from low-salinity surface waters. Light (250 µmol photons m-2 s-1) and anoxia (0 µM O2) stimulated growth and N2 fixation; however, diazotrophy in light was dependent on high organic carbon levels (35 mM, glucose:succinate). Immunolabeling revealed that cellular nitrogenase levels increased with light, decreasing inorganic nitrogen (N) and ambient O2 (250 µM). Light and O2 stimulated motility and biofilm formation on surfaces, and N2 fixation rates increased compared to the control treatment. N2 fixation rates were positively correlated with the formation of rosette-like cellular structures, and an increased concentration of nitrogenase was observed toward the center of these structures, which increased their occurrence 600 times when cultures reached maximum N2 fixation rates vs when they had low rates. Interestingly, N2 fixation was not completely inhibited under oxic conditions and was accompanied by increased formation of capsules and cysts. Rosettes, as well as capsules and cysts, may thus serve as protection against O2. Our study reveals the physiological adaptations that underlie N2 fixation in an anoxygenic phototroph, emphasizing the significance of biofilm formation for utilizing light and fixing N2 under oxic conditions, and underscores the need for deciphering the importance of light for marine NCDs. IMPORTANCE The contribution of non-cyanobacterial diazotrophs (NCDs) to total N2 fixation in the marine water column is unknown, but their importance is likely constrained by the limited availability of dissolved organic matter and low O2 conditions. Light could support N2 fixation and growth by NCDs, yet no examples from bacterioplankton exist. In this study, we show that the phototrophic NCD, Rhodopseudomonas sp. BAL398, which is a member of the diazotrophic community in the surface waters of the Baltic Sea, can utilize light. Our study highlights the significance of biofilm formation for utilizing light and fixing N2 under oxic conditions and the role of cell plasticity in regulating these processes. Our findings have implications for the general understanding of the ecology and importance of NCDs in marine waters. [ABSTRACT FROM AUTHOR]

Cladophora represents a microscopic forest that provides many ecological niches and fosters a diverse microbiota. However, the microbial community on Cladophora in brackish lakes is still poorly understood. In this study, the epiphytic bacterial communities of Cladophora in Qinghai Lake were investigated at three life stages (attached, floating, and decomposing). We found that in the attached stage, Cladophora was enriched with chemoheterotrophic and aerobic microorganisms, including Yoonia-Loktanella and Granulosicoccus. The proportion of phototrophic bacteria was higher in the floating stage, especially Cyanobacteria. The decomposing stage fostered an abundance of bacteria that showed vertical heterogeneity from the surface to the bottom. The surface layer of Cladophora contained mainly stress-tolerant chemoheterotrophic and photoheterotrophic bacteria, including Porphyrobacter and Nonlabens. The microbial community in the middle layer was similar to that of floatingstage Cladophora. Purple oxidizing bacteria were enriched in the bottom layer, with Candidatus Chloroploca, Allochromatium, and Thiocapsa as the dominant genera. The Shannon and Chao1 indices of epibiotic bacterial communities increased monotonically from the attached stage to the decomposing stage. Microbial community composition and functional predictions indicate that a large number of sulfur cycle-associated bacteria play an important role in the development of Cladophora. These results suggest that the microbial assemblage on Cladophora in a brackish lake is complex and contributes to the cycling of materials. IMPORTANCE Cladophora represents a microscopic forest that provides many ecological niches fostering a diverse microbiota, with a complex and intimate relationship between Cladophora and bacteria. Many studies have focused on the microbiology of freshwater Cladophora, but the composition and succession of microorganisms in different life stages of Cladophora, especially in brackish water, have not been explored. In this study, we investigated the microbial assemblages in the life stages of Cladophora in the brackish Qinghai Lake. We show that heterotrophic and photosynthetic autotrophic bacteria are enriched in attached and floating Cladophora, respectively, whereas the epiphytic bacterial community shows vertical heterogeneity in decomposing mats. [ABSTRACT FROM AUTHOR]

Prokaryotes contribute to the global sulfur cycle by using diverse sulfur compounds as sulfur sources or electron acceptors. In this study, we report that a nitrogenase-like enzyme (NFL) and a radical SAM enzyme (RSE) are involved in the novel anaerobic assimilation pathway of a sulfonate, isethionate, in the photosynthetic bacterium Rhodobacter capsulatus . The nflHDK genes for NFL are localized at a locus containing genes for known sulfonate metabolism in the genome. A gene nflB encoding an RSE is present just upstream of nflH , forming a small gene cluster nflBHDK . Mutants lacking any nflBHDK genes are incapable of growing with isethionate as the sole sulfur source under anaerobic photosynthetic conditions, indicating that all four NflBHDK proteins are essential for the isethionate assimilation pathway. Heterologous expression of the islAB genes encoding a known isethionate lyase that degrades isethionate to sulfite and acetaldehyde restored the isethionate-dependent growth of a mutant lacking nflDK , indicating that the enzyme encoding nflBHDK is involved in an isethionate assimilation reaction to release sulfite. Furthermore, the heterologous expression of nflBHDK and ssuCAB encoding an isethionate transporter in the closely related species R. sphaeroides , which does not have nflBHDK and cannot grow with isethionate as the sole sulfur source, conferred isethionate-dependent growth ability to this species. We propose to rename nflBHDK as isrBHDK ( is ethionate r eductase). The isrBHDK genes are widely distributed among various prokaryote phyla. Discovery of the isethionate assimilation pathway by IsrBHDK provides a missing piece for the anaerobic sulfur cycle and for understanding the evolution of ancient sulfur metabolism.IMPORTANCENitrogenase is an important enzyme found in prokaryotes that reduces atmospheric nitrogen to ammonia and plays a fundamental role in the global nitrogen cycle. It has been noted that nitrogenase-like enzymes (NFLs), which share an evolutionary origin with nitrogenase, have evolved to catalyze diverse reactions such as chlorophyll biosynthesis (photosynthesis), coenzyme F 430 biosynthesis (methanogenesis), and methionine biosynthesis. In this study, we discovered that an NFL with unknown function in the photosynthetic bacterium Rhodobacter capsulatus is a novel isethionate reductase (Isr), which catalyzes the assimilatory degradation of isethionate, a sulfonate, releasing sulfite used as the sulfur source under anaerobic conditions. Isr is widely distributed among various bacterial phyla, including intestinal bacteria, and is presumed to play an important role in sulfur metabolism in anaerobic environments such as animal guts and microbial mats. This finding provides a clue for understanding ancient metabolism that evolved under anaerobic environments at the dawn of life., Competing Interests: The authors declare no conflict of interest.

The purple nonsulfur phototrophic bacterium Rhodobacter sphaeroides produces hydrogen gas (H2) from acetate. An approach to improve the H2 production is preventing accumulation of an intracellular energy storage molecule known as poly (b-hydroxybutyrate) (PHB), which competes with H2 production for reducing power. However, disruption of PHB biosynthesis has been reported to severely impair the acetate assimilation depending on the genetic backgrounds and/or culture conditions. To solve this problem, we analyzed the relationship between PHB accumulation and acetate metabolism in R. sphaeroides. Gene deletion analyses based on the wild-type strain revealed that among the two polyhydroxyalkanoate synthase genes in the genome, phaC1, but not phaC2, is essential for PHB accumulation, and the phaC1 deletion mutant exhibited slow growth with acetate. On the other hand, a strain with the deletion of phaC1 together with phaR, which encodes a transcriptional regulator capable of sensing PHB accumulation, exhibited growth comparable to that of the wild-type strain despite no accumulation of PHB. These results suggest that PHB accumulation is required for normal growth with acetate by altering the expression of genes under the control of phaR. This hypothesis was supported by a transcriptome sequencing (RNA-seq) analysis revealing that phaR is involved in the regulation of the ethylmalonyl coenzyme A pathway for acetate assimilation. Consistent with these findings, deletion of phaC1 in a genetically engineered H2-producing strain resulted in lower H2 production from acetate due to growth defects, whereas deletion of phaR together with phaC1 restored growth with acetate and increased H2 production from acetate without PHB accumulation. [ABSTRACT FROM AUTHOR]

The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris strain CGA009 uses the three-carbon dicarboxylic acid malonate as the sole carbon source under phototrophic conditions. However, this bacterium grows extremely slowly on this compound and does not have operons for the two pathways for malonate degradation that have been detected in other bacteria. Many bacteria grow on a spectrum of carbon sources, some of which are classified as poor growth substrates because they support low growth rates. This trait is rarely addressed in the literature, but slow growth is potentially useful in biotechnological applications where it is imperative for bacteria to divert cellular resources to value-added products rather than to growth. This prompted us to explore the genetic and physiological basis for the slow growth of R. palustris with malonate as a carbon source. There are two unlinked genes annotated as encoding a malonyl coenzyme A (malonyl- CoA) synthetase (MatB) and a malonyl-CoA decarboxylase (MatA) in the genome of R. palustris, which we verified as having the predicted functions. Additionally, two tripartite ATP-independent periplasmic transporters (TRAP systems) encoded by rpa2047 to rpa2049 and rpa2541 to rpa2543 were needed for optimal growth on malonate. Most of these genes were expressed constitutively during growth on several carbon sources, including malonate. Our data indicate that R. palustris uses a piecemeal approach to growing on malonate. The data also raise the possibility that this bacterium will evolve to use malonate efficiently if confronted with an appropriate selection pressure. IMPORTANCE There is interest in understanding how bacteria metabolize malonate because this three-carbon dicarboxylic acid can serve as a building block in bioengineering applications to generate useful compounds that have an odd number of carbons. We found that the phototrophic bacterium Rhodopseudomonas palustris grows extremely slowly on malonate. We identified two enzymes and two TRAP transporters involved in the uptake and metabolism of malonate, but some of these elements are apparently not very efficient. R. palustris cells growing with malonate have the potential to be excellent biocatalysts, because cells would be able to divert cellular resources to the production of value-added compounds instead of using them to support rapid growth. In addition, our results suggest that R. palustris is a candidate for directed evolution studies to improve growth on malonate and to observe the kinds of genetic adaptations that occur to make a metabolic pathway operate more efficiently. [ABSTRACT FROM AUTHOR]

Tryptophan (Trp) is an essential aromatic amino acid that has value as an animal feed supplement as the amount found in plant based sources is insufficient. An alternative production by engineered microbial fermentation is to have tryptophan biosynthesized by photosynthetic microorganism that could replace or supplement both the plant and industrially used microbes. We selected Synechocystis sp. PCC 6803, a model cyanobacteria, as the host and studied metabolic engineering and random mutagenesis approaches. Previous work on engineering heterotrophic microbes for improved Trp titers have targeted allosteric feedback regulation in enzymes 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS) and anthranilate synthase (AS) as major bottlenecks in the shikimate pathway. In this work the genes coding feedback resistant enzymes from E.coli, aroGfbr and trpEfbr were overexpressed in the host wild type (WT) strain. Separately, the WT strain was subjected to random mutagenesis and selection using an amino acid analog to isolate tryptophan overproducing strains. The randomly mutagenized strains were sequenced in order to identify the mutations that resulted in the desirable phenotypes. Interestingly, the tryptophan overproducers had mutations in the gene coding chorismate mutase (CM) which catalyzes the conversion of chorismate to prephenate. The best tryptophan overproducer from random mutagenesis was selected as a host for metabolic engineering where aroGfbr and trpEfbr were overexpressed. The best strain developed produced 212±23 mg/L of tryptophan after 10 days of photoautotrophic growth under 3% (v/v) CO2. We demonstrated that a combination of random mutagenesis and metabolic engineering was superior to either individual approach. [ABSTRACT FROM AUTHOR]

Fe-organic matter (Fe-OM) complexes are abundant in the environment and, due to their mobility, reactivity, and bioavailability, play a significant role in the biogeochemical Fe cycle. In photic zones of aquatic environments, Fe-OM complexes can potentially be reduced and oxidized, and thus cycled, by light-dependent processes, including abiotic photoreduction of Fe(III)-OM complexes and microbial oxidation of Fe(II)-OM complexes, by anoxygenic phototrophic bacteria. This could lead to a cryptic iron cycle in which continuous oxidation and rereduction of Fe could result in a low and steady-state Fe(II) concentration despite rapid Fe turnover. However, the coupling of these processes has never been demonstrated experimentally. In this study, we grew a model anoxygenic phototrophic Fe(II) oxidizer, Rhodobacter ferrooxidans SW2, with either citrate, Fe(II)-citrate, or Fe(III)-citrate. We found that strain SW2 was capable of reoxidizing Fe(II)-citrate produced by photochemical reduction of Fe(III)-citrate, which kept the dissolved Fe(II)-citrate concentration at low (<10 μM) and stable concentrations, with a concomitant increase in cell numbers. Cell suspension incubations with strain SW2 showed that it can also oxidize Fe(II)- EDTA, Fe(II)-humic acid, and Fe(II)-fulvic acid complexes. This work demonstrates the potential for active cryptic Fe cycling in the photic zone of anoxic aquatic environments, despite low measurable Fe(II) concentrations which are controlled by the rate of microbial Fe(II) oxidation and the identity of the Fe-OM complexes. [ABSTRACT FROM AUTHOR]

Anoxygenic phototrophic bacteria (APBs) occur in a wide range of aquatic habitats, from hot springs to freshwater lakes and intertidal microbial mats. Here, we report the discovery of a novel niche for APBs: endoliths within marine littoral carbonates. In a study of 40 locations around Isla de Mona, Puerto Rico, and Menorca, Spain, 16S rRNA high-throughput sequencing of endolithic community DNA revealed the presence of abundant phylotypes potentially belonging to wellknown APB clades. An ad hoc phylogenetic classification of these sequences enabled us to refine the assignments more stringently. Even then, all locations contained such putative APBs, often reaching a significant proportion of all phototrophic sequences. In fact, in some 20% of samples, their contribution exceeded that of oxygenic phototrophs, previously regarded as the major type of endolithic microbe in carbonates. The communities contained representatives of APBs in the Chloroflexales, various proteobacterial groups, and Chlorobi. The most abundant phylotypes varied with geography: on Isla de Mona, Roseiflexus and Chlorothrix-related phylotypes dominated, whereas those related to Erythrobacter were the most common in Menorca. The presence of active populations of APBs was corroborated through an analysis of photopigments: bacteriochlorophylls were detected in all samples, bacteriochlorophyll c and a being most abundant. We discuss the potential metabolism and geomicrobial roles of endolithic APBs. Phylogenetic inference suggests that APBs may be playing a role as photoheterotrophs, adding biogeochemical complexity to our understanding of such communities. Given the global extent of coastal carbonate platforms, they likely represent a very large and unexplored habitat for APBs. [ABSTRACT FROM AUTHOR]

The genus Limnohabitans (Comamonadaceae, Betaproteobacteria) is a common and a highly active component of freshwater bacterioplanktonic communities. To date, the genus has been considered to contain only heterotrophic species. In this study, we detected the photosynthesis genes pufLM and bchY in 28 of 46 strains from three Limnohabitans lineages. The pufM sequences obtained are very closely related to environmental pufM sequences detected in various freshwater habitats, indicating the ubiquity and potential importance of photoheterotrophic Limnohabitans in nature. Additionally, we sequenced and analyzed the genomes of 5 potentially photoheterotrophic Limnohabitans strains, to gain further insights into their phototrophic capacity. The structure of the photosynthesis gene cluster turned out to be highly conserved within the genus Limnohabitans and also among all potentially photosynthetic Betaproteobacteria strains. The expression of photosynthetic complexes was detected in a culture of Limnohabitans planktonicus II-D5T using spectroscopic and pigment analyses. This was further verified by a novel combination of infrared microscopy and fluorescent in situ hybridizationIMPORTANCE The data presented document that the capacity to perform anoxygenic photosynthesis is common among the members of the genus Limnohabitans, indicating that they may have a novel role in freshwater habitats. [ABSTRACT FROM AUTHOR]

Terrestrial symbiotic cyanobacteria of the genus Nostoc exhibit a large potential for the production of bioactive natural products of the nonribosomal peptide, polyketide, and ribosomal peptide classes, and yet most of the biosynthetic gene clusters are silent under conventional cultivation conditions. In the present study, we utilized a high-density cultivation approach recently developed for phototrophic bacteria to rapidly generate biomass of the filamentous bacteria up to a density of 400 g (wet weight)/liter. Unexpectedly, integrated transcriptional and metabolomics studies uncovered a major reprogramming of the secondary metabolome of two Nostoc strains at high culture density and a governing effect of extracellular signals in this process. The holistic approach enabled capturing and structural elucidation of novel variants of anabaenopeptin, including one congener with potent allelopathic activity against a strain isolated from the same habitat. The study provides a snapshot on the role of cell-type-specific expression for the formation of natural products in cyanobacteria. [ABSTRACT FROM AUTHOR]

Most described nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB) are mixotrophic and depend on organic cosubstrates for growth. Encrustation of cells in Fe(III) minerals has been observed for mixotrophic NRFeOB but not for autotrophic phototrophic and microaerophilic Fe(II) oxidizers. So far, little is known about cellmineral associations in the few existing autotrophic NRFeOB. Here, we investigate whether the designated autotrophic Fe(II)-oxidizing strain (closely related to Gallionella and Sideroxydans) or the heterotrophic nitrate reducers that are present in the autotrophic nitrate-reducing Fe(II)-oxidizing enrichment culture KS form mineral crusts during Fe(II) oxidation under autotrophic and mixotrophic conditions. In the mixed culture, we found no significant encrustation of any of the cells both during autotrophic oxidation of 8 to 10 mM Fe(II) coupled to nitrate reduction and during cultivation under mixotrophic conditions with 8 to 10 mM Fe(II), 5 mM acetate, and 4 mM nitrate, where higher numbers of heterotrophic nitrate reducers were present. Two pure cultures of heterotrophic nitrate reducers (Nocardioides and Rhodanobacter) isolated from culture KS were analyzed under mixotrophic growth conditions. We found green rust formation, no cell encrustation, and only a few mineral particles on some cell surfaces with 5 mM Fe(II) and some encrustation with 10 mM Fe(II). Our findings suggest that enzymatic, autotrophic Fe(II) oxidation coupled to nitrate reduction forms poorly crystalline Fe(III) oxyhydroxides and proceeds without cellular encrustation while indirect Fe(II) oxidation via heterotrophic nitrate-reductionderived nitrite can lead to green rust as an intermediate mineral and significant cell encrustation. The extent of encrustation caused by indirect Fe(II) oxidation by reactive nitrogen species depends on Fe(II) concentrations and is probably negligible under environmental conditions in most habitats. IMPORTANCE Most described nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB) are mixotrophic (their growth depends on organic cosubstrates) and can become encrusted in Fe(III) minerals. Encrustation is expected to be harmful and poses a threat to cells if it also occurs under environmentally relevant conditions. Nitrite produced during heterotrophic denitrification reacts with Fe(II) abiotically and is probably the reason for encrustation in mixotrophic NRFeOB. Little is known about cell-mineral associations in autotrophic NRFeOB such as the enrichment culture KS. Here, we show that no encrustation occurs in culture KS under autotrophic and mixotrophic conditions while heterotrophic nitrate-reducing isolates from culture KS become encrusted. These findings support the hypothesis that encrustation in mixotrophic cultures is caused by the abiotic reaction of Fe(II) with nitrite and provide evidence that Fe(II) oxidation in culture KS is enzymatic. Furthermore, we show that the extent of encrustation caused by indirect Fe(II) oxidation by reactive nitrogen species depends on Fe(II) concentrations and is probably negligible in most environmental habitats. [ABSTRACT FROM AUTHOR]

Aerobic anoxygenic phototrophic bacteria (AAPB) are thought to be important players in oceanic carbon and energy cycling in the euphotic zone of the ocean. The genus Citromicrobium, widely found in oligotrophic oceans, is a member of marine alphaproteobacterial AAPB. Nine Citromicrobium strains isolated from the South China Sea, the Mediterranean Sea, or the tropical South Atlantic Ocean were found to harbor identical 16S rRNA sequences. The sequencing of their genomes revealed high synteny in major regions. Nine genetic islands (GIs) involved mainly in type IV secretion systems, flagellar biosynthesis, prophage, and integrative conjugative elements, were identified by a fine-scale comparative genomics analysis. These GIs played significant roles in genomic evolution and divergence. Interestingly, the coexistence of two different photosynthetic gene clusters (PGCs) was not only found in the analyzed genomes but also confirmed, for the first time, to our knowledge, in environmental samples. The prevalence of the coexistence of two different PGCs may suggest an adaptation mechanism for Citromicrobium members to survive in the oceans. Comparison of genomic characteristics (e.g., GIs, average nucleotide identity [ANI], single-nucleotide polymorphisms [SNPs], and phylogeny) revealed that strains within a marine region shared a similar evolutionary history that was distinct from that of strains isolated from other regions (South China Sea versus Mediterranean Sea). Geographic differences are partly responsible for driving the observed genomic divergences and allow microbes to evolve through local adaptation. Three Citromicrobium strains isolated from the Mediterranean Sea diverged millions of years ago from other strains and evolved into a novel group. [ABSTRACT FROM AUTHOR]

Microbial sulfur metabolism, particularly the formation and consumption of insoluble elemental sulfur (S0), is an important biogeochemical engine that has been harnessed for applications ranging from bioleaching and biomining to remediation of waste streams. Chlorobaculum tepidum, a low-light-adapted photoautolithotrophic sulfur-oxidizing bacterium, oxidizes multiple sulfur species and displays a preference for more reduced electron donors: sulfide>S0>thiosulfate. To understand this preference in the context of light energy availability, an "energy landscape" of phototrophic sulfur oxidation was constructed by varying electron donor identity, light flux, and culture duration. Biomass and cellular parameters of C. tepidum cultures grown across this landscape were analyzed. From these data, a correction factor for colorimetric protein assays was developed, enabling more accurate biomass measurements for C. tepidum, as well as other organisms. C. tepidum's bulk amino acid composition correlated with energy landscape parameters, including a tendency toward less energetically expensive amino acids under reduced light flux. This correlation, paired with an observation of increased cell size and storage carbon production under electron- rich growth conditions, suggests that C. tepidum has evolved to cope with changing energy availability by tuning its proteome for energetic efficiency and storing compounds for leaner times. [ABSTRACT FROM AUTHOR]

The use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3= end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest. [ABSTRACT FROM AUTHOR]

Anoxygenic phototrophs represent an environmentally important and phylogenetically diverse group of organisms. They harvest light using bacteriochlorophyll-containing reaction centers. Recently, a novel phototrophic bacterium, Gemmatimonas phototrophica, belonging to a rarely studied phylum, Gemmatimonadetes, was isolated from a freshwater lake in the Gobi Desert. To obtain more information about the environmental distribution of phototrophic Gemmatimonadetes, we collected microbial samples from the water column, upper sediment, and deeper anoxic sediment of Lake Taihu, China. MiSeq sequencing of the 16S rRNA, pufM, and bchY genes was carried out to assess the diversity of local phototrophic communities. In addition, we designed new degenerate primers of aerobic cyclase gene acsF, which serves as a convenient marker for both phototrophic Gemmatimonadetes and phototrophic Proteobacteria. Our results showed that most of the phototrophic species in Lake Taihu belong to Alpha- and Betaproteobacteria. Sequences of green sulfur and green nonsulfur bacteria (phototrophic Chlorobi and Chloroflexi, respectively) were found in the sediment. Using the newly designed primers, we identified a diverse community of phototrophic Gemmatimonadetes forming 30 operational taxonomic units. These species represented 10.5 and 17.3% of the acsF reads in the upper semiaerobic sediment and anoxic sediment, whereas their abundance in the water column was<1%. [ABSTRACT FROM AUTHOR]

Microbes can produce molecular hydrogen (H2) via fermentation, dinitrogen fixation, or direct photolysis, yet the H2 dynamics in cyanobacterial communities has only been explored in a few natural systems and mostly in the laboratory. In this study, we investigated the diel in situ H2 dynamics in a hot spring microbial mat, where various ecotypes of unicellular cyanobacteria (Synechococcus sp.) are the only oxygenic phototrophs. In the evening, H2 accumulated rapidly after the onset of darkness, reaching peak values of up to 30µmol H2 liter-1 at about 1-mm depth below the mat surface, slowly decreasing to about 11 µmol H2 liter-1 just before sunrise. Another pulse of H2 production, reaching a peak concentration of 46 µmol H2 liter-1, was found in the early morning under dim light conditions too low to induce accumulation of O2 in the mat. The light stimulation of H2 accumulation indicated that nitrogenase activity was an important source of H2 during the morning. This is in accordance with earlier findings of a distinct early morning peak in N2 fixation and expression of Synechococcus nitrogenase genes in mat samples from the same location. Fermentation might have contributed to the formation of H2 during the night, where accumulation of other fermentation products lowered the pH in the mat to less than pH 6 compared to a spring source pH of 8.3. [ABSTRACT FROM AUTHOR]

Bacteriophytochrome photoreceptors (BphPs) and their cognate response regulators make up two-component signal transduction systems which direct bacteria to mount phenotypic responses to changes in environmental light quality. Most of these systems utilize single-domain response regulators to transduce signals through unknown pathways and mechanisms. Here we describe the photocycle and autophosphorylation kinetics of RtBphP1, a red light-regulated histidine kinase from the desert bacterium Ramlibacter tataouinensis. RtBphP1 undergoes red to far-red photoconversion with rapid thermal reversion to the dark state. RtBphP1 is autophosphorylated in the dark; this activity is inhibited under red light. The RtBphP1 cognate response regulator, the R. tataouinensis bacteriophytochrome response regulator (RtBRR), and a homolog, AtBRR from Agrobacterium tumefaciens, crystallize unexpectedly as arm-in-arm dimers, reliant on a conserved hydrophobic motif, hFWAhL (where h is a hydrophobic M, V, L, or I residue). RtBRR and AtBRR dimerize distinctly from four structurally characterized phytochrome response regulators found in photosynthetic organisms and from all other receiver domain homodimers in the Protein Data Bank. A unique cacodylate-zinc-histidine tag metal organic framework yielded single-wavelength anomalous diffraction phases and may be of general interest. Examination of the effect of the BRR stoichiometry on signal transduction showed that phosphorylated RtBRR is accumulated more efficiently than the engineered monomeric RtBRR (RtBRRmon) in phosphotransfer reactions. Thus, we conclude that arm-in-arm dimers are a relevant signaling intermediate in this class of two-component regulatory systems. [ABSTRACT FROM AUTHOR]

[NiFe]-hydrogenases are regulated by various factors to fulfill their physiological functions in bacterial cells. The photosynthetic purple sulfur bacterium Thiocapsa roseopersicina harbors four functional [NiFe]-hydrogenases: HynSL, HupSL, Hox1, and Hox2. Most of these hydrogenases are functionally linked to sulfur metabolism, and thiosulfate has a central role in this organism. The membrane-associated Hup hydrogenases have been shown to play a role in energy conservation through hydrogen recycling. The expression of Hup-type hydrogenases is regulated by H2 in Rhodobacter capsulatus and Cupriavidus necator; however, it has been shown that the corresponding hydrogen-sensing system is nonfunctional in T. roseopersicina and that thiosulfate is a regulating factor of hup expression. Here, we describe the discovery and analysis of mutants of a putative regulator (HupO) of the Hup hydrogenase in T. roseopersicina. HupO appears to mediate the transcriptional repression of Hup enzyme synthesis under low-thiosulfate conditions. We also demonstrate that the presence of the Hox1 hydrogenase strongly influences Hup enzyme synthesis in that hup expression was decreased significantly in the hox1 mutant. This reduction in Hup synthesis could be reversed by mutation of hupO, which resulted in strongly elevated hup expression, as well as Hup protein levels, and concomitant in vivo hydrogen uptake activity in the hox1 mutant. However, this regulatory control was observed only at low thiosulfate concentrations. Additionally, weak hydrogen-dependent hup expression was shown in the hupO mutant strain lacking the Hox1 hydrogenase. HupO-mediated Hup regulation therefore appears to link thiosulfate metabolism and the hydrogenase network in T. roseopersicina. [ABSTRACT FROM AUTHOR]

Iron is abundant in sediments, where it can be biogeochemically cycled between its divalent and trivalent redox states. The neutrophilic microbiological Fe cycle involves Fe(III)-reducing and three different physiological groups of Fe(II)-oxidizing microorganisms, i.e., microaerophilic, anoxygenic phototrophic, and nitrate-reducing Fe(II) oxidizers. However, it is unknown whether all three groups coexist in one habitat and how they are spatially distributed in relation to gradients of O2, light, nitrate, and Fe(II). We examined two coastal marine sediments in Aarhus Bay, Denmark, by cultivation and most probable number (MPN) studies for Fe(II) oxidizers and Fe(III) reducers and by quantitative-PCR (qPCR) assays for microaerophilic Fe(II) oxidizers. Our results demonstrate the coexistence of all three metabolic types of Fe(II) oxidizers and Fe(III) reducers. In qPCR, microaerophilic Fe(II) oxidizers (Zetaproteobacteria) were present with up to 3.2 × 106 cells g dry sediment-1. In MPNs, nitrate-reducing Fe(II) oxidizers, anoxygenic phototrophic Fe(II) oxidizers, and Fe(III) reducers reached cell numbers of up to 3.5 × 104, 3.1 × 102, and 4.4 × 104 g dry sediment-1, respectively. O2 and light penetrated only a few millimeters, but the depth distribution of the different iron metabolizers did not correlate with the profile of O2, Fe(II), or light. Instead, abundances were homogeneous within the upper 3 cm of the sediment, probably due to wave-induced sediment reworking and bioturbation. In microaerophilic Fe(II)-oxidizing enrichment cultures, strains belonging to the Zetaproteobacteria were identified. Photoferrotrophic enrichments contained strains related to Chlorobium and Rhodobacter; the nitrate-reducing Fe(II) enrichments contained strains related to Hoeflea and Denitromonas. This study shows the coexistence of all three types of Fe(II) oxidizers in two near-shore marine environments and the potential for competition and interrelationships between them. [ABSTRACT FROM AUTHOR]

Microbial sulfide oxidation in aquatic environments is an important ecosystem process, as sulfide is potently toxic to aerobic organisms. Sulfide oxidation in anoxic waters can prevent the efflux of sulfide to aerobic water masses, thus mitigating toxicity. The contribution of phototrophic sulfide-oxidizing bacteria to anaerobic sulfide oxidation in the Chesapeake Bay and the redox chemistry of the stratified water column were investigated in the summers of 2011 to 2014. In 2011 and 2013, phototrophic sulfide-oxidizing bacteria closely related to Prosthecochloris species of the phylum Chlorobi were cultivated from waters sampled at and below the oxic-anoxic interface, where measured light penetration was sufficient to support populations of low-light-adapted photosynthetic bacteria. In 2012, 2013, and 2014, light-dependent sulfide loss was observed in freshly collected water column samples. In these samples, extremely low light levels caused 2- to 10-fold increases in the sulfide uptake rate over the sulfide uptake rate under dark conditions. An enrichment, CB11, dominated by Prosthecochloris species, oxidized sulfide with a Ks value of 11 μM and a Vmax value of 51 μM min-1 (mg protein-1). Using these kinetic values with in situ sulfide concentrations and light fluxes, we calculated that a small population of Chlorobi similar to those in enrichment CB11 can account for the observed anaerobic light-dependent sulfide consumption activity in natural water samples. We conclude that Chlorobi play a far larger role in the Chesapeake Bay than currently appreciated. This result has potential implications for coastal anoxic waters and expanding oxygen-minimum zones as they begin to impinge on the photic zone. [ABSTRACT FROM AUTHOR]

Most stratified sulfidic holomictic lakes become oxygenated after annual turnover. In contrast, Lake Rogoznica, on the eastern Adriatic coast, has been observed to undergo a period of water column anoxia after water layer mixing and establishment of holomictic conditions. Although Lake Rogoznica's chemistry and hydrography have been studied extensively, it is unclear how the microbial communities typically inhabiting the oxic epilimnion and a sulfidic hypolimnion respond to such a drastic shift in redox conditions. We investigated the impact of anoxic holomixis on microbial diversity and microbially mediated sulfur cycling in Lake Rogoznica with an array of culture-independent microbiological methods. Our data suggest a tight coupling between the lake's chemistry and occurring microorganisms. During stratification, anoxygenic phototrophic sulfur bacteria were dominant at the chemocline and in the hypolimnion. After an anoxic mixing event, the anoxygenic phototrophic sulfur bacteria entirely disappeared, and the homogeneous, anoxic water column was dominated by a bloom of gammaproteobacterial sulfur oxidizers related to the GSO/SUP05 clade. This study is the first report of a community shift from phototrophic to chemotrophic sulfide oxidizers as a response to anoxic holomictic conditions in a seasonally stratified seawater lake. [ABSTRACT FROM AUTHOR]

The purple sulfur bacterium Allochromatium vinosum DSM 180T is one of the best-studied sulfur-oxidizing anoxygenic phototrophic bacteria, and it has been developed into a model organism for laboratory-based studies of oxidative sulfur metabolism. Here, we took advantage of the organism's high metabolic versatility and performed whole-genome transcriptional profiling to investigate the response of A. vinosum cells upon exposure to sulfide, thiosulfate, elemental sulfur, or sulfite compared to photoorganoheterotrophic growth on malate. Differential expression of 1,178 genes was observed, corresponding to 30% of the A. vinosum genome. Relative transcription of 551 genes increased significantly during growth on one of the different sulfur sources, while the relative transcript abundance of 627 genes decreased. A significant number of genes that revealed strongly enhanced relative transcription levels have documented sulfur metabolism-related functions. Among these are the dsr genes, including dsrAB for dissimilatory sulfite reductase, and the sgp genes for the proteins of the sulfur globule envelope, thus confirming former results. In addition, we identified new genes encoding proteins with appropriate subcellular localization and properties to participate in oxidative dissimilatory sulfur metabolism. Those four genes for hypothetical proteins that exhibited the strongest increases of mRNA levels on sulfide and elemental sulfur, respectively, were chosen for inactivation and phenotypic analyses of the respective mutant strains. This approach verified the importance of the encoded proteins for sulfur globule formation during the oxidation of sulfide and thiosulfate and thereby also documented the suitability of comparative transcriptomics for the identification of new sulfur-related genes in anoxygenic phototrophic sulfur bacteria. [ABSTRACT FROM AUTHOR]

In their natural environment, bacteria often behave differently than they do under laboratory conditions. To gain insight into the physiology of bacteria in situ, dedicated approaches are required to monitor their adaptations and specific behaviors under environmental conditions. Optical microscopy is crucial for the observation of fundamental characteristics of bacteria, such as cell shape, size, and marker gene expression. Here, fluidic force microscopy (FluidFM) was exploited to isolate optically selected bacteria for subsequent identification and characterization. In this study, bacteriochlorophyll-producing bacteria, which can be visualized due to their characteristic fluorescence in the infrared range, were isolated from leaf washes. Bacterial communities from the phyllosphere were investigated because they harbor genes indicative of aerobic anoxygenic photosynthesis. Our data show that different species of Methylobacterium express their photosystem inplanta, and they show a distinct pattern of bacteri-ochlorophyll production under laboratory conditions that is dependent on supplied carbon sources. [ABSTRACT FROM AUTHOR]

Magnesium-protoporphyrin IX monomethylester cyclase is one of the key enzymes of the bacteriochlorophyll biosynthesis pathway. There exist two fundamentally different forms of this enzyme. The oxygen-dependent form, encoded by the gene acsF, catalyzes the formation of the bacteriochlorophyll fifth ring using oxygen, whereas the oxygen-independent form encoded by the genebchE utilizes an oxygen atom extracted from water. The presence of acsF andbchE genes was surveyed in various phototrophic Proteobacteria using the available genomic data and newly designed degenerated primers. It was found that while the majority of purple nonsulfur bacteria contained both forms of the cyclase, the purple sulfur bacteria contained only the oxygen-independent form. All tested species of aerobic anoxygenic phototrophs contained acsF genes, but some of them also retained the bchE gene. In contrast to bchE phylogeny, theacsF phylogeny was in good agreement with 16S inferred phylogeny. Moreover, the survey of the genome data documented that the acsF gene occupies a conserved position inside the photosynthesis gene cluster, whereas the bchElocation in the genome varied largely between the species. This suggests that the oxygen-dependent cyclase was recruited by purple phototrophic bacteria very early during their evolution. The primary sequence and immunochemical similarity with its cyanobacterial counterparts suggests that acsF may have been acquired by Proteobacteria via horizontal gene transfer from cyanobacteria. The acquisition of the gene allowed purple nonsulfur phototrophic bacteria to proliferate in the mildly oxygenated conditions of the Proterozoic era. [ABSTRACT FROM AUTHOR]

The ability of photosynthetic Bradyrhizobium strains 0RS285 and 0RS278 to nodulate soybeans was investigated. While the nod gene-deficient 0RS278 strain induced bumps only on soybean roots, the nod gene-containing 0RS285 strain formed nitrogen-fixing nodules. However, symbiotic efficiencies differed drastically depending on both the soybean genotype used and the culture conditions tested. [ABSTRACT FROM AUTHOR]

Environmental gradients are expected to promote the diversification and coexistence of ecological specialists adapted to local conditions. Consistent with this view, genera of phototrophic microorganisms in alkaline geothermal systems generally appear to consist of anciently divergent populations which have specialized on different temperature habitats. At White Creek (Lower Geyser Basin, Yellowstone National Park), however, a novel, 16S rRNA-defined lineage of the filamentous anoxygenic phototroph Chioroflexus (OTU 10, phylum Chloroflexi) occupies a much wider thermal niche than other 16S rRNA-defined groups of phototrophic bacteria. This suggests that Chioroflexus OTU lOis either an ecological generalist or, alternatively, a group of cryptic thermal specialists which have recently diverged. To distinguish between these alternatives, we first isolated laboratory strains of Chioroflexus OTU 10 from along the White Creek temperature gradient. These strains are identical for partial gene sequences encoding the 16S rRNA and malonyl coenzyme A (CoA) reductase. However, strains isolated from upstream and downstream samples could be distinguished based on sequence variation at pcs, which encodes the propionyl-CoA synthase of the 3-hydroxypropionate pathway of carbon fixation used by the genus Chloroflexus. We next demonstrated that strains have diverged in temperature range for growth. Specifically, we obtained evidence for a positive correlation between thermal niche breadth and temperature optimum, with strains isolated from lower temperatures exhibiting greater thermal specialization than the most thermotolerant strain. The study has implications for our understanding of both the process of niche diversification of microorganisms and how diversity is organized in these hot spring communities. [ABSTRACT FROM AUTHOR]

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxicanoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 pm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes. [ABSTRACT FROM AUTHOR]

Aerobic anoxygenic phototrophs contain photosynthetic reaction centers composed of bacteriochiorophyll. These organisms are photoheterotrophs, as they require organic carbon substrates for their growth whereas light-derived energy has only an auxiliary function. To establish the contribution of light energy to their metabolism, we grew the phototrophic strain Erythrobacter sp. NAP1 in a carbon-limited chemostat regimen on defined carbon sources (glutamate, pyruvate, acetate, and glucose) under con- ditions of different light intensities. When grown in a light-dark cycle, these bacteria accumulated 25% to 110% more biomass in terms of carbon than cultures grown in the dark. Cultures grown on glutamate accumulated the most biomass at moderate light intensities of 50 to 150 μmol m-2 s-1 but were inhibited at higher light intensities. In the case of pyruvate, we did not find any inhibition of growth by high irradiance. The extent of anaplerotic carbon fixation was detemined by radioactive bicarbonate incorporation assays. While the carboxylation activity provided 4% to 11% of the cellular carbon in the pyruvate-grown culture, in the glutamate-grown cells it provided only approximately 1% of the carbon. Additionally, we tested the effect of light on respiration and photosynthetic electron flow. With increasing light intensity, respiration decreased to approximately 25% of its dark value and was replaced by photophosphorylation. The additional energy from light allows the aerobic anoxygenic phototrophs to accumulate the supplied organic carbon which would otherwise be respired. The higher efficiency of organic carbon utilization may provide an important competitive advantage during growth under carbon-limited conditions. [ABSTRACT FROM AUTHOR]

Aerobic anoxygenic phototrophic (AAP) bacteria are photoheterotrophic microbes that are found in a broad range of aquatic environments. Although potentially significant to the microbial ecology and biogeochemistry of marine ecosystems, their abundance and genetic diversity and the environmental variables that regulate these properties are poorly understood. Using samples along nearshore/offshore transects from five disparate islands in the Pacific Ocean (Oahu, Molokai, Futuna, Aniwa, and Lord Howe) and off California, we show that AAP bacteria, as quantified by the pufM gene biomarker, are most abundant near shore and in areas with high chlorophyll or Synechococcus abundance. These AAP bacterial populations are genetically diverse, with most members belonging to the alpha- or gammaproteobacterial groups and with subclades that are associated with specific environmental variables. The genetic diversity of AAP bacteria is structured along the nearshore/offshore transects in relation to environmental variables, and uncultured pufM gene libraries suggest that nearshore communities are distinct from those offshore. AAP bacterial communities are also genetically distinct between islands, such that the stations that are most distantly separated are the most genetically distinct. Together, these results demonstrate that environmental variables regulate both the abundance and diversity of AAP bacteria but that endemism may also be a contributing factor in structuring these communities. [ABSTRACT FROM AUTHOR]

Enterococcus species composition was determined each hour for 72 h at a polluted marine beach in Avalon, Santa dataiina Island, CA. Species composition during the day was significantly different from that at night, based on an analysis of similarity. Enterococcusfaecium and E. faecalis were more prevalent at night than during the day, while E. hirae and other Enterococcus species were more prevalent during the day than the night. Entero coccus spp. containing a yellow pigment were more common during the day than the night, suggesting that the pigmented phenotype may offer a competitive advantage under sunlit conditions. A laboratory microcosm experiment established that the pigmented E. casseliflavus isolate and a pigmented. faecalis isolate recovered from the field site decay slower than a nonpigmented E. faecalis isolate in a solar simulator in simulated, clear seawater. This further supports the idea that the yellow carotenoid pigment in Enterococcus provides protection under sunlit conditions. The findings are in accordance with previous work with other carotenoid-containing nonphotosynthetic and photosynthetic bacteria that suggests that the carotenoid is able to quench reactive oxygen species capable of causing ph9toinactivation and photostress. The results suggest that using enterococcal species composition as a microbial source tracking tool may be hindered by the differential environmental persistence of pigmented and nonpigmented enterococci. [ABSTRACT FROM AUTHOR]

Recent physiological work has shown that the filamentous euendolithic cyanobacterium Mastigocoleus testarum (strain BC008) is able to bore into solid carbonates using Ca2+-ATPases to take up Ca2+ from the medium at the excavation front, promoting dissolution of CaCO3 there. It is not known, however, if this is a widespread mechanism or, rather, a unique capability of this model strain. To test this, we undertook a survey of multispecies euendolithic microbial assemblages infesting natural carbonate substrates in marine coastal waters of the Caribbean, Mediterranean, South Pacific, and Sea of Cortez. Microscopic examination revealed the presence of complex assemblages of euendoliths, encompassing 3 out of the 5 major cyanobacterial orders. 16S rRNA gene clone libraries detected even greater diversity, particularly among the thin-filamentous forms, and allowed us to categorize the endoliths in our samples into 8 distinct phylogenetic clades. Using real-time Ca2+ imaging under a confocal laser scanning microscope, we could show that all communities displayed light-dependent formation of Ca2+-supersaturated zones in and around boreholes, a staple of actively boring phototrophs. In 3 out of 4 samples, boring activity was sensitive to at least one of two inhibitors of Ca2+-ATPase transporters (thapsigargin or tert-butylhydroquinone), indicating that the Ca2+-ATPase mechanism is widespread among cyanobacterial euendoliths but perhaps not universal. Function-community structure correlations point to one particular clade of baeocyte-forming euendoliths as the potential exception. [ABSTRACT FROM AUTHOR]

Growth is one of the basic attributes of any living organism. Surprisingly, the growth rates of marine bacterioplankton are only poorly known. Current data suggest that marine bacteria grow relatively slowly, having generation times of several days. However, some bacterial groups, such as the aerobic anoxygenic phototrophic (AAP) bacteria, have been shown to grow much faster. Two manipulation experiments, in which grazing, viruses, and resource competition were reduced, were conducted in the coastal Mediterranean Sea (Blanes Bay Microbial Observatory). The growth rates of AAP bacteria and of several important phylogenetic groups (the Bacteroidetes, the aiphaproteobacterial groups Roseobacter and SAR11, and the Gammaproteobacteria group and its subgroups the Alteromonadaceae and the NOR5/0M60 dade) were calculated from changes in cell numbers in the manipulation treatments. In addition, we examined the role that top-down (mortality due to grazers and viruses) and bottom-up (resource availability) factors play in determining the growth rates of these groups. Manipulations resulted in an increase of the growth rates of all groups studied, but its extent differed largely among the individual treatments and among the different groups. Interestingly, higher growth rates were found for the AAP bacteria (up to 3.71 day1) and for the Alteromonadaceae (up to 5.44 day'), in spite of the fact that these bacterial groups represented only a very low percentage of the total prokaryotic community. In contrast, the SARi! dade, which was the most abundant group, was the slower grower in all treatments. Our results show that, in general, the least abundant groups exhibited the highest rates, whereas the most abundant groups were those growing more slowly, indicating that some minor groups, such the AAP bacteria, very likely contribute much more to the recycling of organic matter in the ocean than what their abundances alone would predict. [ABSTRACT FROM AUTHOR]

Aerobic anoxygenic phototrophic bacteria (AAPB) are unique players in carbon cycling in the ocean. Cellular carbon storage is an important mechanism regulating the nutrition status of AAPB but is not yet well understood. In this paper, six AAPB species (Dinoroseobacter sp. JL1447, Roseobacter denitrificans OCh 114, Roseobacter litoralis OCh 149, Dinoroseobacter shibae DFL 12T, Labrenzia alexandrii DFL 11T, and Erythrobacter longus DSMZ 6997) were examined, and all of them demonstrated the ability to form the carbon polymer polyhydroxyalkanoate (PHA) in the cell. The PHA in Dinoroseobacter sp. JL1447 was identified as poly-betahydroxybutyrate (PHB) according to evidence from Fourier transform infrared spectroscopy, differential scanning calorimetry, and `H nuclear magnetic resonance spectroscopy examinations. Carbon sources turned out to be critical for PHA production in AAPB. Among the eight media tested with Dinoroseobacter sp. JL 1447, sodium acetate, giving a PIIA production rate of 72%, was the most productive carbon source, followed by glucose, with a 68% PIIA production rate. Such PHA production rates are among the highest recorded for all bacteria. The C/N ratio of substrates was verified by the experiments as another key factor in PHA production. In the case of R. denitrificans OCh 114, PHA was not detected when the organism was cultured at C/N ratios of <2 but became apparent at C/N ratios of >3. Light is also important for the formation of PHA in AAPB. In the case of Dinoroseobacter sp. JL1447, up to a one-quarter increase in PHB production was observed when the culture underwent growth in a light-dark cycle compared to growth completely in the dark. [ABSTRACT FROM AUTHOR]

Over the past quarter-century, microbiologists have used DNA sequence information to aid in the characterization of microbial communities. During the last decade, this has expanded from single genes to microbial community genomics, or metagenomics, in which the gene content of an environment can provide not just a census of the community members but direct information on metabolic capabilities and potential interactions among community members. Here we introduce a method for the quantitative characterization and comparison of microbial communities based on the normalization of metagenomic data by estimating average genome sizes. This normalization can relieve comparative biases introduced by differences in community structure, number of sequencing reads, and sequencing read lengths between different metagenomes. We demonstrate the utility of this approach by comparing metagenomes from two different marine sources using both conventional small-subunit (SSU) rRNA gene analyses and our quantitative method to calculate the proportion of genomes in each sample that are capable of a particular metabolic trait. With both environments, to determine what proportion of each community they make up and how differences in environment affect their abundances, we characterize three different types of autotrophic organisms: aerobic, photosynthetic carbon fixers (the Cyanobacteria); anaerobic, photosynthetic carbon fixers (the Chlorobi); and anaerobic, nonphotosynthetic carbon fixers (the Desulfobacteraceae). These analyses demonstrate how genome proportionality compares to SSU rRNA gene relative abundance and how factors such as average genome size and SSU rRNA gene copy number affect sampling probability and therefore both types of community analysis. [ABSTRACT FROM AUTHOR]

Light is a ubiquitous environmental signal that many organisms sense and respond to by modulating their physiological responses accordingly. While this is an expected response among phototrophic microorganisms, the ability of chemotrophic prokaryotes to sense and react to light has become a puzzling and novel issue in bacterial physiology, particularly among bacterial pathogens. In this work, we show that the opportunistic pathogen Acinetobacter baumannii senses and responds to blue light. Motility and formation of biofilms and pellicles were observed only when bacterial cells were incubated in darkness. In contrast, the killing of Candida albicans filaments was enhanced when they were cocultured with bacteria under light. These bacterial responses depend on the expression of the A. baumannii ATCC 17978 A1S&lowbar;2225 gene, which codes for an 18.6-kDa protein that contains an N-terminal blue-light-sensing-using flavin (BLUF) domain and lacks a detectable output domain(s). Spectral analyses of the purified recombinant protein showed its ability to sense light by a red shift upon illumination. Therefore, the A1S&lowbar;2225 gene, which is present in several members of the Acinetobacter genus, was named blue-light-sensing A (blsA). Interestingly, temperature plays a role in the ability of A. baumannii to sense and respond to light via the BlsA photoreceptor protein. [ABSTRACT FROM AUTHOR]

A challenge for photobiological production of hydrogen gas (H2) as a potential biofuel is to find suitable electron-donating feedstocks. Here, we examined the inorganic compound thiosulfate as a possible electron donor for nitrogenase-catalyzed H2 production by the purple nonsulfur phototrophic bacterium (PNSB) Rhodopseudomonas palustris. Thiosulfate is an intermediate of microbial sulfur metabolism in nature and is also generated in industrial processes. We found that R. palustris grew photoautotrophically with thiosulfate and bicarbonate and produced H2 when nitrogen gas was the sole nitrogen source (nitrogen-fixing conditions). In addition, illuminated nongrowing R. palustris cells converted about 80% of available electrons from thiosulfate to H2∙ H2 production with acetate and succinate as electron donors was less efficient (40 to 60%), partly because nongrowing cells excreted the intermediary metabolite α-ketoglutarate into the culture medium. The fixABCX operon (RPA4602 to RPA4605) encoding a predicted electron-transfer complex is necessary for growth using thiosulfate under nitrogen-fixing conditions and may serve as a point of engineering to control rates of H2 production. The possibility to use thiosulfate expands the range of electron-donating compounds for H2 production by PNSBs beyond biomass-based electron donors. [ABSTRACT FROM AUTHOR]

Natural noncoding small RNAs have been shown to be involved in a number of cellular processes as regulators. Using the mechanisms thus elucidated, artificial small interfering RNAs (siRNAs), ribozymes, and RNA aptamers are also expected to be potential candidates for RNA therapeutic agents. However, current techniques are too costly for industrial production of these RNAs for use as drugs. Here, we propose a new method for in vivo production of artificial RNAs using the marine phototrophic bacterium Rhodovulum sulfidophilum. Using engineered plasmids and this bacterium, which produces extracellular nucleic acids in nature, we developed a method for extracellular production of a streptavidin RNA aptamer. As the bacterium does not produce any RNases in the culture medium, at least within the cultivation period tested, the designed RNA itself is produced and retained in the culture medium of the bacterium without any specific mechanism for protection against degradation by nucleases. Here, we report that the streptavidin RNA aptamer is produced in the culture medium and retains its specific function. This is the first demonstration of extracellular production of a functional artificial RNA in vivo, which will pave the way for inexpensive production of RNA drugs. [ABSTRACT FROM AUTHOR]

Recent studies highlight the diversity and significance of marine phototrophic microorganisms such as picocyanobacteria, phototrophic picoeukaryotes, and bacteriochlorophyll- and rhodopsin-holding phototrophic bacteria. To assess if freshwater ecosystems also harbor similar phototroph diversity, genes involved in the biosynthesis of bacteriochlorophyll and chlorophyll were targeted to explore oxygenic and aerobic anoxygenic phototroph composition in a wide range of lakes. Partial dark-operative protochlorophyllide oxidoreductase (DPOR) and chlorophyllide oxidoreductase (COR) genes in bacteria of seven lakes with contrasting trophic statuses were PCR amplified, cloned, and sequenced. Out of 61 sequences encoding the L subunit of DPOR (L-DPOR), 22 clustered with aerobic anoxygenic photosynthetic bacteria, whereas 39 L-DPOR sequences related to oxygenic phototrophs, like cyanobacteria, were observed. Phylogenetic analysis revealed clear separation of these freshwater L-DPOR genes as well as 11 COR gene sequences from their marine counterparts. Terminal restriction fragment length analysis of L-DPOR genes was used to characterize oxygenic aerobic and anoxygenic photosynthesizing populations in 20 lakes differing in physical and chemical characteristics. Significant differences in L-DPOR community composition were observed between dystrophic lakes and all other systems, where a higher proportion of genes affiliated with aerobic anoxygenic photosynthetic bacteria was observed than in other systems. Our results reveal a significant diversity of phototrophic microorganisms in lakes and suggest niche partitioning of oxygenic and aerobic anoxygenic phototrophs in these systems in response to trophic status and coupled differences in light regime. [ABSTRACT FROM AUTHOR]

Diverse bacteria are known to oxidize millimolar concentrations of ferrous iron [Fe(II)] under anaerobic conditions, both phototrophically and chemotrophically. Yet whether they can do this under conditions that are relevant to natural systems is understood less well. In this study, we tested how light, Fe(II) speciation, pH, and salinity affected the rate of Fe(II) oxidation by Rhodobacter capsulatus SB1003. Although R. eapsulatus cannot grow photoautotrophically on Fe(II), it oxidizes Fe(II) at rates comparable to those of bacteria that do grow photoautotrophically on Fe(II) as soon as it is exposed to light, provided it has a functional photosystem. Chelation of Fe(II) by diverse organic ligands promotes Fe(H) oxidation, and as the pH increases, so does the oxidation rate, except in the presence of nitrilotriacetate; nonchelated forms of Fe(II) are also more rapidly oxidized at higher pH. Salt concentrations typical of marine environments inhibit Fe(II) oxidation. When growing photoheterotrophically on humic substances, R. capsulatus is highly sensitive to low concentrations of Fe(II); it is inhibited in the presence of concentrations as low as 5 p~M. The product of Fe(II) oxidation, ferric iron, does not hamper growth under these conditions. When other parameters, such as pH or the presence of chelators, are adjusted to promote Fe(II) oxidation, the growth inhibition effect of Fe(II) is alleviated. Together, these results suggest that Fe(II) is toxic to R. capsulatus growing under strictly anaerobic conditions and that Fe(II) oxidation alleviates this toxicity. [ABSTRACT FROM AUTHOR]

Nitrogenase activity in Rhodospirillum rubrum and in some other photosynthetic bacteria is regulated in part by the availability of light. This regulation is through a posttranslational modification system that is itself regulated by PII homologs in the cell. PII is one of the most broadly distributed regulatory proteins in nature and directly or indirectly senses nitrogen and carbon signals in the cell. However, its possible role in responding to light availability remains unclear. Because PII binds ATP, we tested the hypothesis that removal of light would affect PII by changing intracellular ATP levels, and this in turn would affect the regulation of nitrogenase activity. This in vivo test involved a variety of different methods for the measurement of ATP, as well as the deliberate perturbation of intracellular ATP levels by chemical and genetic means. To our surprise, we found fairly normal levels of nitrogenase activity and posttranslational regulation of nitrogenase even under conditions of drastically reduced ATP levels. This indicates that low ATP levels have no more than a modest impact on the PII-mediated regulation of NifA activity and on the posttranslational regulation of nitrogenase activity. The relatively high nitrogenase activity also shows that the ATP-dependent electron flux from dinitrogenase reductase to dinitrogenase is also surprisingly insensitive to a depleted ATP level. These in vivo results disprove the simple model of ATP as the key energy signal to PII under these conditions. We currently suppose that the ratio of ADP/ATP might be the relevant signal, as suggested by a number of recent in vitro analyses. [ABSTRACT FROM AUTHOR]

Photoheterotrophic microbes, which are capable of utilizing dissolved organic materials and harvesting light energy, include coccoid cyanobacteria (Synechococcus and Prochiorococcus), aerobic anoxygenic phototrophic (AAP) bacteria, and proteorhodopsin (PR)-containing bacteria. Our knowledge of photoheterotrophic mi- crobes is largely incomplete, especially for high-latitude waters such as the Arctic Ocean, where photohetero- trophs may have special ecological relationships and distinct biogeochemical impacts due to extremes in day length and seasonal ice cover. These microbes were examined by epifluorescence microscopy, flow cytometry, and quantitative PCR (QPCR) assays for PR and a gene diagnostic of AAP bacteria (puJM). The abundance of AAP bacteria and PR-containing bacteria decreased from summer to winter, in parallel with a threefold decrease in the total prokaryotic community. In contrast, the abundance of Synechococcus organisms did not decrease in winter, suggesting that their growth was supported by organic substrates. Results from QPCR assays revealed no substantial shifts in the community structure of AAP bacteria and PR-containing bacteria. However, Arctic PR genes were different from those found at lower latitudes, and surprisingly, they were not similar to those in Antarctic coastal waters. Photoheterotrophic microbes appear to compete successfully with strict heterotrophs during winter darkness below the ice, but AAP bacteria and PR-containing bacteria do not behave as superior competitors during the summer. [ABSTRACT FROM AUTHOR]

Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus numerically dominate the picophytoplankton of the world ocean, making a key contribution to global primaly production. Prochiorococcus was isolated around 20 years ago and is probably the most abundant photosynthetic organism on Earth. The genus comprises specific ecolypes which are phylogenetically distinct and differ markedly in their photophysiology, allowing growth over a broad range of light and nutrient conditions within the 45°N to 40°S latitudinal belt that they occupy. Synechococcus and Prochlorococcus are closely related, together forming a discrete picophytoplankton dade, but are distinguishable by their possession of dissimilar light- harvesting apparatuses and differences in cell size and elemental composition. Synechococcus strains have a ubiquitous oceanic distribution compared to that of Prochlorococcus strains and are characterized byphylogenetically discrete lineages with a wide range ofpigmentation. in this review, we put our current knowledge of marine picocyanobacterial genomics into an environmental context and present previously unpublished genomic information arising from extensive genonhic comparisons in order to provide insights into the adaptations of these marine microbes to their environment and how they are reflected at the genomic level. [ABSTRACT FROM AUTHOR]