Genomic Evolution of the Marine Bacterium Phaeobacter inhibens during Biofilm Growth.

Phaeobacter inhibens 2.10 is an effective biofilm former on marine surfaces and has the ability to outcompete other microorganisms, possibly due to the production of the plasmid-encoded secondary metabolite tropodithietic acid (TDA). P. inhibens 2.10 biofilms produce phenotypic variants with reduced competitiveness compared to the wild type. In the present study, we used longitudinal, genome-wide deep sequencing to uncover the genetic foundation that contributes to the emergent phenotypic diversity in P. inhibens 2.10 biofilm dispersants. Our results show that phenotypic variation is not due to the loss of the plasmid that carries the genes for TDA synthesis but instead show that P. inhibens 2.10 biofilm populations become rapidly enriched in single nucleotide variations in genes involved in the synthesis of TDA. While variants in genes previously linked to other phenotypes, such as lipopolysaccharide production (i.e., rfbA) and cellular persistence (i.e., metG), also appear to be selected for during biofilm dispersal, the number and consistency of variations found for genes involved in TDA production suggest that this metabolite imposes a burden on P. inhibens 2.10 cells. Our results indicate a strong selection pressure for the loss of TDA in monospecies biofilm populations and provide insight into how competition (or a lack thereof) in biofilms might shape genome evolution in bacteria. IMPORTANCE Biofilm formation and dispersal are important survival strategies for environmental bacteria. During biofilm dispersal, cells often display stable and heritable variants from the parental biofilm. Phaeobacter inhibens is an effective colonizer of marine surfaces, in which a subpopulation of its biofilm dispersal cells displays a noncompetitive phenotype. This study aimed to elucidate the genetic basis of these phenotypic changes. Despite the progress made to date in characterizing the dispersal variants in P. inhibens, little is understood about the underlying genetic changes that result in the development of the specific variants. Here, P. inhibens phenotypic variation was linked to single nucleotide polymorphisms (SNPs), in particular in genes affecting the competitive ability of P. inhibens, including genes related to the production of the antibiotic tropodithietic acid (TDA) and bacterial cell-cell communication (e.g., quorum sensing). This work is significant as it reveals how the biofilm lifestyle might shape genome evolution in a cosmopolitan bacterium.

Salibaculum halophilum gen. nov., sp. nov. and Salibaculum griseiflavum sp. nov., in the family Rhodobacteraceae.

Two Gram-stain-negative, moderately halophilic, non-motile, rod-shaped, pale yellow, and aerobic strains, designated WDS1C4T and WDS4C29T, were isolated from a marine solar saltern in Weihai, Shandong Province, PR China. Growth of strain WDS1C4T occurred at 10-45 °C (optimum, 37 °C), with 4-16 % (w/v) NaCl (optimum, 8 %) and at pH 6.5-9.0 (optimum, pH 7.5). Growth of strain WDS4C29T occurred at 10-45 °C (optimum, 40 °C), with 2-18 % (w/v) NaCl (optimum, 6 %) and at pH 6.5-9.0 (optimum, pH 7.5). Q-10 was the sole respiratory quinone of the two strains. The major polar lipids of strains WDS1C4T and WDS4C29T were phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The major cellular fatty acid in strains WDS1C4T and WDS4C29T was C18 : 1 ω7c, and the genomic DNA G+C contents of strains WDS1C4T and WDS4C29T were 67.6 and 63.3 mol%, respectively. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strains WDS1C4T and WDS4C29T were members of the family Rhodobacteraceae and showed 94.3 and 95.3 % similarities to their closest relative, Celeribacter indicus, respectively. The similarity between WDS1C4T and WDS4C29T was 97.3 %. Differential phenotypic and genotypic characteristics of the two isolates from recognized genera showed that the two strains should be classified as representing two novel species in a new genus for which the names Salibaculum halophilum gen. nov., sp. nov. (type species, type strain WDS1C4T=MCCC 1H00179T=KCTC 52542T) and Salibaculum griseiflavum sp. nov. (WDS4C29T=MCCC 1H00175T=KCTC 52541T) are proposed.

Potential risks of microplastics combined with superbugs: Enrichment of antibiotic resistant bacteria on the surface of microplastics in mariculture system.

Microplastics have become emerging pollutants and served as potential vectors for harmful bacteria, while rare information on the emergency and propagation of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) on the surface of microplastics is available. This study investigated the enrichment of ARB, especially multi-antibiotic resistant bacteria (MARB), on the surface of microplastics in mariculture system. Polyethylene terephthalate accounted for the highest proportion (75%) in the collected microplastics. The counts of cultivable ARB in microplastic samples were 6.40 × 106-2.48 × 108 cfu/g, which were 100-5000 times higher than those in water samples. The ratios of cultivable ARB to total cultivable bacteria from microplastic samples were higher than those from water samples. High-throughput sequencing showed that the diversity and abundance of cultivable ARB in the microplastic samples was high with the predominant bacterial genera of Vibrio, Muricauda and Ruegeria. Total 160 MARB isolates were obtained and most of isolates were obtained from the microplastic samples. MARB isolates resisting or intermediating to four and three antibiotics accounted for much higher proportions in the microplastic samples, and the higher percentage of antibiotic resistance was to penicillin, sulfafurazole, erythromycin and tetracycline. The dominant multiple antibiotic resistance profile was TET-SFX-ERY-PEN, which accounted for 25.4% in microplastic samples and 23.9% in water samples. In typical MARB isolates, the positive detection rate of ARGs was up to 80.0% in microplastic samples while that was 65.3% in water samples. Five types of class 1 integrons (intI1) associated gene cassette arrays and seven types of gene cassettes were detected in microplastic samples, which were more than those in water samples. These results revealed that microplastics were hazardous pollutants for the enrichment of ARB, especially superbugs, and the spread of antibiotic resistance.

Amino Acid and Sugar Catabolism in the Marine Bacterium Phaeobacter inhibens DSM 17395 from an Energetic Viewpoint.

Growth energetics and metabolic efficiency contribute to the lifestyle and habitat imprint of microorganisms. Roseobacters constitute one of the most abundant and successful marine bacterioplankton groups. Here, we reflect on the energetics and metabolic efficiency of Phaeobacter inhibens DSM 17395, a versatile heterotrophic roseobacter. Fourteen different substrates (five sugars and nine amino acids) and their degradation pathways were assessed for energetic efficiencies based on catabolic ATP yields, calculated from net formed ATP and reducing equivalents. The latter were converted into ATP by employing the most divergent coupling ratios (i.e., ions per ATP) currently known for F1Fo ATP synthases in heterotrophic bacteria. The catabolic ATP yields of the pathways studied in P. inhibens differed ∼3-fold. The actual free energy costs for ATP synthesis were estimated at 81.6 kJ per mol ATP (3.3 ions per ATP) or 104.2 kJ per mol ATP (4.3 ions per ATP), yielding an average thermodynamic efficiency of ∼37.7% or ∼29.5%, respectively. Growth performance (rates, yields) and carbon assimilation efficiency were determined for P. inhibens growing in process-controlled bioreactors with 10 different single substrates (Glc, Man, N-acetylglucosamine [Nag], Phe, Trp, His, Lys, Thr, Val, or Leu) and with 2 defined substrate mixtures. The efficiencies of carbon assimilation into biomass ranged from ∼28% to 61%, with His/Trp and Thr/Leu yielding the lowest and highest levels. These efficiencies correlated with catabolic and ATP yields only to some extent. Substrate-specific metabolic demands and/or functions, as well as the compositions of the substrate mixtures, apparently affected the energetic costs of growth. These include energetic burdens associated with, e.g., slow growth, stress, and/or the production of tropodithietic acid.IMPORTANCE Heterotrophic members of the bacterioplankton serve the marine ecosystem by transforming organic matter, an activity that is governed by the bacterial growth efficiencies (BGEs) obtained under given environmental conditions. In marine ecology, the concept of BGE refers to the carbon assimilation efficiency within natural communities. The marine bacterium studied here, Phaeobacter inhibens DSM 17395, is a copiotrophic representative of the globally abundant Roseobacter group, and the 15 catabolic pathways investigated are widespread among these marine heterotrophs. Combining pathway-specific catabolic ATP yields with in-depth quantitative physiological data could (i) provide a new baseline for the study of growth energetics and efficiency in further Roseobacter group members and other copiotrophic marine bacteria in productive coastal ecosystems and (ii) contribute to a better understanding of the factors controlling BGE (including the additional energetic burden arising from widespread secondary-metabolite formation) based on laboratory studies with pure cultures.

Role of RpoN from Labrenzia aggregata LZB033 (Rhodobacteraceae) in Formation of Flagella and Biofilms, Motility, and Environmental Adaptation.

Labrenzia aggregata LZB033 (Rhodobacteraceae), which produces dimethylsulfoniopropionate (DMSP) and reduces nitrate to nitrogen, was isolated from seawater of the East China Sea. Its genome encodes a large number of transcriptional regulators which may be important for its adaptation to diverse marine environments. The alternative σ54 factor (RpoN) is a central regulator of many bacteria, regulating the transcription of multiple genes and controlling important cellular functions. However, the exact role of RpoN in Labrenzia spp. is unknown. In this study, an in-frame rpoN deletion mutant was constructed in LZB033, and the function of RpoN was determined. To systematically identify RpoN-controlled genes, we performed a detailed analysis of gene expression differences between the wild-type strain and the ΔrpoN mutant using RNA sequencing. The expression of 175 genes was shown to be controlled by RpoN. Subsequent phenotypic assays showed that the ΔrpoN mutant was attenuated in flagellar biosynthesis and swimming motility, utilized up to 13 carbon substrates differently, lacked the ability to assimilate malic acid, and displayed markedly decreased biofilm formation. In addition, stress response assays showed that the ΔrpoN mutant was impaired in the ability to survive under different challenge conditions, including osmotic stress, oxidative stress, temperature changes, and acid stress. Moreover, both the DMSP synthesis and catabolism rates of LZB033 decreased after rpoN was knocked out. Our work provides essential insight into the regulatory function of RpoN, revealing that RpoN is a key determinant for LZB033 flagellar formation, motility, biofilm formation, and environmental fitness, as well as DMSP production and degradation.IMPORTANCE This study established an in-frame gene deletion method in the alphaproteobacterium Labrenzia aggregata LZB033 and generated an rpoN gene mutant. A comparison of the transcriptomes and phenotypic characteristics between the mutant and wild-type strains confirmed the role of RpoN in L. aggregata LZB033 flagellar formation, motility, biofilm formation, and carbon usage. Most importantly, RpoN is a key factor for survival under different environmental challenge conditions. Furthermore, the ability to synthesize and metabolize dimethylsulfoniopropionate (DMSP) was related to RpoN. These features revealed RpoN to be an important regulator of stress resistance and survival for L. aggregata LZB033 in marine environments.

Synthetic cell-cell communication in a three-species consortium for one-step vitamin C fermentation.

OBJECTIVES: A three-species consortium for one-step fermentation of 2-keto-L-gulonic acid (2-KGA) was constructed to better strengthen the cell-cell communication. And the programmed cell death module based on the LuxI/LuxR quorum-sensing (QS) system was established in Gluconobacter oxydans to reduce the competition that between G. oxydans and Ketogulonicigenium vulgare. RESULTS: By constructing and optimizing the core region of the promoter, which directly regulated the expression of lethal ccdB genes in QS system, IR3C achieved the best lethal effect. The consortium of IR3C- K. vulgare-Bacillus megaterium (abbreviated as 3C) achieved the highest 2-KGA titer (68.80 ± 4.18 g/l), and the molar conversion rate was 80.7% within 36 h in 5 l fermenter. Metabolomic analysis on intracellular small molecules of consortia 3C and 1C showed that most amino acids (such as glycine, leucine, methionine and proline) and TCA cycle intermediates (such as succinic acid, fumaric acid and malic acid) were significantly affected. These results further validated that the programmed cell death module based on the LuxI/LuxR QS system in G. oxydans could also faciliate better growth and higher production of consortium 3C for one-step fermentation. CONCLUSIONS: We successfully constructed a novel three-species consortia for one-step vitamin C fermentation by strengthening the cell-cell communication. This will be very useful for probing the rational design principles of more complex multi-microbial consortia.

Heme and nitric oxide binding by the transcriptional regulator DnrF from the marine bacterium Dinoroseobacter shibae increases napD promoter affinity.

Under oxygen-limiting conditions, the marine bacterium Dinoroseobacter shibae DFL12T generates energy via denitrification, a respiratory process in which nitric oxide (NO) is an intermediate. Accumulation of NO may cause cytotoxic effects. The response to this nitrosative (NO-triggered) stress is controlled by the Crp/Fnr-type transcriptional regulator DnrF. We analyzed the response to NO and the mechanism of NO sensing by the DnrF regulator. Using reporter gene fusions and transcriptomics, here we report that DnrF selectively repressed nitrate reductase (nap) genes, preventing further NO formation. In addition, DnrF induced the expression of the NO reductase genes (norCB), which promote NO consumption. We used UV-visible and EPR spectroscopy to characterize heme binding to DnrF and subsequent NO coordination. DnrF detects NO via its bound heme cofactor. We found that the dimeric DnrF bound one molecule of heme per subunit. Purified recombinant apo-DnrF bound its target promoter sequences (napD, nosR2, norC, hemA, and dnrE) in electromobility shift assays, and we identified a specific palindromic DNA-binding site 5'-TTGATN4ATCAA-3' in these target sequences via mutagenesis studies. Most importantly, successive addition of heme as well as heme and NO to purified recombinant apo-DnrF protein increased affinity of the holo-DnrF for its specific binding motif in the napD promoter. On the basis of these results, we propose a model for the DnrF-mediated NO stress response of this marine bacterium.

Ecology and Biotechnological Potential of Bacteria Belonging to the Genus Pseudovibrio.

Members of the genus Pseudovibrio have been isolated worldwide from a great variety of marine sources as both free-living and host-associated bacteria. So far, the available data depict a group of alphaproteobacteria characterized by a versatile metabolism, which allows them to use a variety of substrates to meet their carbon, nitrogen, sulfur, and phosphorous requirements. Additionally, Pseudovibrio-related bacteria have been shown to proliferate under extreme oligotrophic conditions, tolerate high heavy-metal concentrations, and metabolize potentially toxic compounds. Considering this versatility, it is not surprising that they have been detected from temperate to tropical regions and are often the most abundant isolates obtained from marine invertebrates. Such an association is particularly recurrent with marine sponges and corals, animals that play a key role in benthic marine systems. The data so far available indicate that these bacteria are mainly beneficial to the host, and besides being involved in major nutrient cycles, they could provide the host with both vitamins/cofactors and protection from potential pathogens via the synthesis of antimicrobial secondary metabolites. In fact, the biosynthetic abilities of Pseudovibrio spp. have been emerging in recent years, and both genomic and analytic studies have underlined how these organisms promise novel natural products of biotechnological value.

Recognition cascade and metabolite transfer in a marine bacteria-phytoplankton model system.

The trophic linkage between marine bacteria and phytoplankton in the surface ocean is a key step in the global carbon cycle, with almost half of marine primary production transformed by heterotrophic bacterioplankton within hours to weeks of fixation. Early studies conceptualized this link as the passive addition and removal of organic compounds from a shared seawater reservoir. Here, we analysed transcript and intracellular metabolite patterns in a two-member model system and found that the presence of a heterotrophic bacterium induced a potential recognition cascade in a marine phytoplankton species that parallels better-understood vascular plant response systems. Bacterium Ruegeria pomeroyi DSS-3 triggered differential expression of >80 genes in diatom Thalassiosira pseudonana CCMP1335 that are homologs to those used by plants to recognize external stimuli, including proteins putatively involved in leucine-rich repeat recognition activity, second messenger production and protein kinase cascades. Co-cultured diatoms also downregulated lipid biosynthesis genes and upregulated chitin metabolism genes. From differential expression of bacterial transporter systems, we hypothesize that nine diatom metabolites supported the majority of bacterial growth, among them sulfonates, sugar derivatives and organic nitrogen compounds. Similar recognition responses and metabolic linkages as observed in this model system may influence carbon transformations by ocean plankton.

Testing the metabolic theory of ecology with marine bacteria: different temperature sensitivity of major phylogenetic groups during the spring phytoplankton bloom.

Although temperature is a key driver of bacterioplankton metabolism, the effect of ocean warming on different bacterial phylogenetic groups remains unclear. Here, we conducted monthly short-term incubations with natural coastal bacterial communities over an annual cycle to test the effect of experimental temperature on the growth rates and carrying capacities of four phylogenetic groups: SAR11, Rhodobacteraceae, Gammaproteobacteria and Bacteroidetes. SAR11 was the most abundant group year-round as analysed by CARD-FISH, with maximum abundances in summer, while the other taxa peaked in spring. All groups, including SAR11, showed high temperature-sensitivity of growth rates and/or carrying capacities in spring, under phytoplankton bloom or post-bloom conditions. In that season, Rhodobacteraceae showed the strongest temperature response in growth rates, estimated here as activation energy (E, 1.43 eV), suggesting an advantage to outcompete other groups under warmer conditions. In summer E values were in general lower than 0.65 eV, the value predicted by the Metabolic Theory of Ecology (MTE). Contrary to MTE predictions, carrying capacity tended to increase with warming for all bacterial groups. Our analysis confirms that resource availability is key when addressing the temperature response of heterotrophic bacterioplankton. We further show that even under nutrient-sufficient conditions, warming differentially affected distinct bacterioplankton taxa.

Native plasmids restrict growth of Phaeobacter inhibens DSM 17395: Energetic costs of plasmids assessed by quantitative physiological analyses.

Plasmid carriage is associated with energetic costs, and thus only those plasmids providing fitness benefits are stably maintained in the host lineage. Marine bacteria of the Roseobacter clade harbor up to 11 extrachromosomal replicons, adding lifestyle-relevant and possibly habitat success-promoting functions to their genomic repertoire. Phaeobacter inhibens DSM 17395 is a nutritionally versatile representative, carrying three stable and functionally distinct plasmids (65, 78, and 262 kb). The present study investigates the physiological and energetic consequences of plasmid carriage in P. inhibens DSM 17395, employing mutants cured from all native plasmids in every possible combination (seven different). Cultivation in process-controlled bioreactors with casamino acids as organic substrate revealed a complex physiological response, suggesting existence of functional interconnections between the replicons. Deletion of the 262 kb plasmid boosted growth rate (>3-fold) and growth efficiency (yields for carbon, O2 and CO2 ), which was not observed for the 65 or 78 kb plasmid. Carriage of the 262 kb plasmid was most costly for the wild type, i.e. contributing ∼50% to its energetic (dissimilatory) expenditures. Cost-benefit analysis of plasmid carriage reflects the high value of plasmids for niche specialization of P. inhibens DSM 17395 and most likely also for related Phaeobacter species.

Phaeobacter inhibens as biocontrol agent against Vibrio vulnificus in oyster models.

Molluscan shellfish can cause food borne diseases and here we investigated if addition of Vibrio-antagonising bacteria could reduce Vibrio vulnificus in model oyster systems and prevent its establishment in live animals. Phaeobacter inhibens, which produces an antibacterial compound, tropodithietic acid (TDA), inhibited V. vulnificus as did pure TDA (MIC of 1-3.9 µM). P. inhibens DSM 17395 (at 10(6) cfu/ml) eradicated 10(5) cfu/ml V. vulnificus CMCP6 (a rifampicin resistant variant) from a co-culture oyster model system (oyster juice) whereas the pathogen grew to 10(7) cfu/ml when co-cultured with a TDA negative Phaeobacter mutant. P. inhibens grew well in oyster juice to 10(8) CFU/ml and sterile filtered samples from these cultures were inhibitory to Vibrio spp. P. inhibens established itself in live European flat oysters (Ostrea edulis) and remained at 10(5) cfu/g for five days. However, the presence of P. inhibens could not prevent subsequently added V. vulnificus from entering the live animals, likely because of too low levels of the biocontrol strain. Whilst the oyster model studies provided indication that P. inhibens DSM 17395 could be a good candidate as biocontrol agent against V. vulnificus further optimization is need in the actual animal rearing situation.

Bacterial community shift is induced by dynamic environmental parameters in a changing coastal ecosystem (northern Adriatic, northeastern Mediterranean Sea)--a 2-year time-series study.

The potential link between the microbial dynamics and the environmental parameters was investigated in a semi-enclosed and highly dynamic coastal system (Gulf of Trieste, northern Adriatic Sea, NE Mediterranean Sea). Our comprehensive 2-year time-series study showed that despite the shallowness of this area, there was a significant difference between the surface and the bottom bacterial community structure. The bottom bacterial community was more diverse than the surface one and influenced by sediment re-suspension. The surface seawater temperature had a profound effect on bacterial productivity, while the bacterial community structure was more affected by freshwater-borne nutrients and phytoplankton blooms. Phytoplankton blooms caused an increase of Gammaproteobacteria (Alteromonadaceae, SAR86 and Vibrionaceae) and shift in dominance from SAR11 to Rhodobacteraceae taxon at the surface. Our results propose the importance of the water mass movements as drivers of freshwater-borne nutrients and of allochthonous microbial taxa. This study emphasizes the prediction power based on association networks analyses that are fed with long-term measurements of microbial and environmental parameters. These interaction maps offer valuable insights into the response of marine ecosystem to climate- and anthropogenic-driven stressors.

Phage-bacteria network analysis and its implication for the understanding of coral disease.

Multiple studies have explored microbial shifts in diseased or stressed corals; however, little is known about bacteriophage interactions with microbes in this context. This study characterized microbial 16S rRNA amplicons and phage metagenomes associated with Montastraea annularis corals during a concurrent white plague disease outbreak and bleaching event. Phage consortia differed between bleached and diseased tissues. Phages in the family Inoviridae were elevated in diseased or healthy tissues compared with bleached portions of diseased tissues. Microbial communities also differed between diseased and bleached corals. Bacteria in the orders Rhodobacterales and Campylobacterales were increased while Kiloniellales was decreased in diseased compared with other tissues. A network of phage-bacteria interactions was constructed of all phage strains and 11 bacterial genera that differed across health states. Phage-bacteria interactions varied in specificity: phages interacted with one to eight bacterial hosts while bacteria interacted with up to 59 phages. Six phages were identified that interacted exclusively with Rhodobacterales and Campylobacterales. These results suggest that phages have a role in controlling stress-associated bacteria, and that networks can be utilized to select potential phages for mitigating detrimental bacterial growth in phage therapy applications.

Phaeobacter sp. strain Y4I utilizes two separate cell-to-cell communication systems to regulate production of the antimicrobial indigoidine.

The marine roseobacter Phaeobacter sp. strain Y4I synthesizes the blue antimicrobial secondary metabolite indigoidine when grown in a biofilm or on agar plates. Prior studies suggested that indigoidine production may be, in part, regulated by cell-to-cell communication systems. Phaeobacter sp. strain Y4I possesses two luxR and luxI homologous N-acyl-L-homoserine lactone (AHL)-mediated cell-to-cell communication systems, designated pgaRI and phaRI. We show here that Y4I produces two dominantAHLs, the novel monounsaturated N-(3-hydroxydodecenoyl)-L-homoserine lactone (3OHC(12:1)-HSL) and the relatively common N-octanoyl-L-homoserine lactone (C8-HSL), and provide evidence that they are synthesized by PhaI and PgaI, respectively.A Tn5 insertional mutation in either genetic locus results in the abolishment (pgaR::Tn5) or reduction (phaR::Tn5) of pigment production. Motility defects and denser biofilms were also observed in these mutant backgrounds, suggesting an overlap in the functional roles of these systems. Production of the AHLs occurs at distinct points during growth on an agar surface and was determined by isotope dilution high-performance liquid chromatography­tandem mass spectrometry (ID-HPLC-MS/MS) analysis.Within 2 h of surface inoculation, only 3OHC(12:1)-HSL was detected in agar extracts. As surface-attached cells became established (at approximately 10 h), the concentration of 3OHC(12:1)-HSL decreased, and the concentration of C8-HSL increased rapidly over 14 h.After longer (>24-h) establishment periods, the concentrations of the two AHLs increased to and stabilized at approximately 15 nM and approximately 600 nM for 3OHC12:1-HSL and C8-HSL, respectively. In contrast, the total amount of indigoidine increased steadily from undetectable to 642 Mby 48 h. Gene expression profiles of the AHL and indigoidine synthases (pgaI, phaI, and igiD) were consistent with their metabolite profiles. These data provide evidence that pgaRI and phaRI play overlapping roles in the regulation of indigoidine biosynthesis, and it is postulated that this allows Phaeobacter sp. strain Y4I to coordinate production of indigoidine with different growth-phase-dependent physiologies.

Structure and function of a short LOV protein from the marine phototrophic bacterium Dinoroseobacter shibae.

BACKGROUND: Light, oxygen, voltage (LOV) domains are widely distributed in plants, algae, fungi, bacteria, and represent the photo-responsive domains of various blue-light photoreceptor proteins. Their photocycle involves the blue-light triggered adduct formation between the C(4a) atom of a non-covalently bound flavin chromophore and the sulfur atom of a conserved cysteine in the LOV sensor domain. LOV proteins show considerable variation in the structure of N- and C-terminal elements which flank the LOV core domain, as well as in the lifetime of the adduct state. RESULTS: Here, we report the photochemical, structural and functional characterization of DsLOV, a LOV protein from the photoheterotrophic marine α-proteobacterium Dinoroseobacter shibae which exhibits an average adduct state lifetime of 9.6 s at 20°C, and thus represents the fastest reverting bacterial LOV protein reported so far. Mutational analysis in D. shibae revealed a unique role of DsLOV in controlling the induction of photopigment synthesis in the absence of blue-light. The dark state crystal structure of DsLOV determined at 1.5 Å resolution reveals a conserved core domain with an extended N-terminal cap. The dimer interface in the crystal structure forms a unique network of hydrogen bonds involving residues of the N-terminus and the ß-scaffold of the core domain. The structure of photoexcited DsLOV suggests increased flexibility in the N-cap region and a significant shift in the Cα backbone of ß strands in the N- and C-terminal ends of the LOV core domain. CONCLUSIONS: The results presented here cover the characterization of the unusual short LOV protein DsLOV from Dinoroseobacter shibae including its regulatory function, extremely fast dark recovery and an N-terminus mediated dimer interface. Due to its unique photophysical, structural and regulatory properties, DsLOV might thus serve as an alternative model system for studying light perception by LOV proteins and physiological responses in bacteria.

L-sorbose is not only a substrate for 2-keto-L-gulonic acid production in the artificial microbial ecosystem of two strains mixed fermentation.

The co-culture system of the fermentation process of vitamin C can be regarded as an artificial microbial ecosystem (AME). To extend our understanding of this AME, an investigation of the relationship between strains, substrate and product was carried out in this study. The results showed that both Ketogulonicigenium vulgare and 2-keto-L-gulonic acid (2-KLG, the precursor of vitamin C) can inhibit the growth of the helper strain, while the helper strain promoted the growth of K. vulgare and 2-KLG production. Moreover, L-sorbose is not only a substrate for 2-KLG production in the AME, but also a promoter of K. vulgare and an inhibitor of the helper strain. In the earlier stage of fermentation, the inhibition of L-sorbose on the helper strain's growth is a key factor for ensuring an efficient fermentation. In the condition of adding the extra helper strain (OD: 0.57, ratio of inoculation: 2%), the yields of 2-KLG is increased by 9% in the 14% L-sorbose medium. To the best of our knowledge, this is the first report about the inhibition of substrate in the AME of 2-KLG production.

Modeling and parameters identification of 2-keto-L-gulonic acid fed-batch fermentation.

This article presents a modeling approach for industrial 2-keto-L-gulonic acid (2-KGA) fed-batch fermentation by the mixed culture of Ketogulonicigenium vulgare (K. vulgare) and Bacillus megaterium (B. megaterium). A macrokinetic model of K. vulgare is constructed based on the simplified metabolic pathways. The reaction rates obtained from the macrokinetic model are then coupled into a bioreactor model such that the relationship between substrate feeding rates and the main state variables, e.g., the concentrations of the biomass, substrate and product, is constructed. A differential evolution algorithm using the Lozi map as the random number generator is utilized to perform the model parameters identification, with the industrial data of 2-KGA fed-batch fermentation. Validation results demonstrate that the model simulations of substrate and product concentrations are well in coincidence with the measurements. Furthermore, the model simulations of biomass concentrations reflect principally the growth kinetics of the two microbes in the mixed culture.

The CtrA phosphorelay integrates differentiation and communication in the marine alphaproteobacterium Dinoroseobacter shibae.

BACKGROUND: Dinoroseobacter shibae, a member of the Roseobacter clade abundant in marine environments, maintains morphological heterogeneity throughout growth, with small cells dividing by binary fission and large cells dividing by budding from one or both cell poles. This morphological heterogeneity is lost if the quorum sensing (QS) system is silenced, concurrent with a decreased expression of the CtrA phosphorelay, a regulatory system conserved in Alphaproteobacteria and the master regulator of the Caulobacter crescentus cell cycle. It consists of the sensor histidine kinase CckA, the phosphotransferase ChpT and the transcriptional regulator CtrA. Here we tested if the QS induced differentiation of D. shibae is mediated by the CtrA phosphorelay. RESULTS: Mutants for ctrA, chpT and cckA showed almost homogeneous cell morphology and divided by binary fission. For ctrA and chpT, expression in trans on a plasmid caused the fraction of cells containing more than two chromosome equivalents to increase above wild-type level, indicating that gene copy number directly controls chromosome number. Transcriptome analysis revealed that CtrA is a master regulator for flagellar biosynthesis and has a great influence on the transition to stationary phase. Interestingly, the expression of the autoinducer synthase genes luxI2 and luxI3 was strongly reduced in all three mutants, resulting in loss of biosynthesis of acylated homoserine-lactones with C14 side-chain, but could be restored by expressing these genes in trans. Several phylogenetic clusters of Alphaproteobacteria revealed a CtrA binding site in the promoters of QS genes, including Roseobacters and Rhizobia. CONCLUSIONS: The CtrA phosphorelay induces differentiation of a marine Roseobacter strain that is strikingly different from that of C. crescentus. Instead of a tightly regulated cell cycle and a switch between two morphotypes, the morphology and cell division of Dinoroseobacter shibae are highly heterogeneous. We discovered for the first time that the CtrA phosphorelay controls the biosynthesis of signaling molecules. Thus cell-cell communication and differentiation are interlinked in this organism. This may be a common strategy, since we found a similar genetic set-up in other species in the ecologically relevant group of Alphaproteobacteria. D. shibae will be a valuable model organism to study bacterial differentiation into pleomorphic cells.

Metabolism of dimethylsulphoniopropionate by Ruegeria pomeroyi†DSS-3.

Ruegeria pomeroyi DSS-3 possesses two general pathways for metabolism of dimethylsulphoniopropionate (DMSP), an osmolyte of algae and abundant carbon source for marine bacteria. In the DMSP cleavage pathway, acrylate is transformed into acryloyl-CoA by propionate-CoA ligase (SPO2934) and other unidentified acyl-CoA ligases. Acryloyl-CoA is then reduced to propionyl-CoA by AcuI or SPO1914. Acryloyl-CoA is also rapidly hydrated to 3-hydroxypropionyl-CoA by acryloyl-CoA hydratase (SPO0147). A SPO1914 mutant was unable to grow on acrylate as the sole carbon source, supporting its role in this pathway. Similarly, growth on methylmercaptopropionate, the first intermediate of the DMSP demethylation pathway, was severely inhibited by a mutation in the gene encoding crotonyl-CoA carboxylase/reductase, demonstrating that acetate produced by this pathway was metabolized by the ethylmalonyl-CoA pathway. Amino acids and nucleosides from cells grown on (13) C-enriched DMSP possessed labelling patterns that were consistent with carbon from DMSP being metabolized by both the ethylmalonyl-CoA and acrylate pathways as well as a role for pyruvate dehydrogenase. This latter conclusion was supported by the phenotype of a pdh mutant, which grew poorly on electron-rich substrates. Additionally, label from [(13) C-methyl] DMSP only appeared in carbons derived from methyl-tetrahydrofolate, and there was no evidence for a serine cycle of C-1 assimilation.