Integrating pH into the metabolic theory of ecology to predict bacterial diversity in soil.

Microorganisms play essential roles in soil ecosystem functioning and maintenance, but methods are currently lacking for quantitative assessments of the mechanisms underlying microbial diversity patterns observed across disparate systems and scales. Here we established a quantitative model to incorporate pH into metabolic theory to capture and explain some of the unexplained variation in the relationship between temperature and soil bacterial diversity. We then tested and validated our newly developed models across multiple scales of ecological organization. At the species level, we modeled the diversification rate of the model bacterium Pseudomonas fluorescens evolving under laboratory media gradients varying in temperature and pH. At the community level, we modeled patterns of bacterial communities in paddy soils across a continental scale, which included natural gradients of pH and temperature. Last, we further extended our model at a global scale by integrating a meta-analysis comprising 870 soils collected worldwide from a wide range of ecosystems. Our results were robust in consistently predicting the distributional patterns of bacterial diversity across soil temperature and pH gradients-with model variation explaining from 7 to 66% of the variation in bacterial diversity, depending on the scale and system complexity. Together, our study represents a nexus point for the integration of soil bacterial diversity and quantitative models with the potential to be used at distinct spatiotemporal scales. By mechanistically representing pH into metabolic theory, our study enhances our capacity to explain and predict the patterns of bacterial diversity and functioning under current or future climate change scenarios.

Role of a local transcription factor in governing cellular carbon/nitrogen homeostasis in Pseudomonas fluorescens.

Autoactivation of two-component systems (TCSs) can increase the sensitivity to signals but inherently cause a delayed response. Here, we describe a unique negative feedback mechanism enabling the global NtrB/NtrC regulator to rapidly respond to nitrogen starvation over the course of histidine utilization (hut) in Pseudomonas fluorescens. NtrBC directly activates transcription of hut genes, but overexpression will produce excess ammonium leading to NtrBC inactivation. To prevent this from occurring, the histidine-responsive repressor HutC fine-tunes ntrBC autoactivation: HutC and NtrC bind to the same operator site in the ntrBC promoter. This newly discovered low-affinity binding site shows little sequence similarity with the consensus sequence that HutC recognizes for substrate-specific induction of hut operons. A combination of genetic and transcriptomic analysis indicated that both ntrBC and hut promoter activities cannot be stably maintained in the ΔhutC background when histidine fluctuates at high concentrations. Moreover, the global carbon regulator CbrA/CbrB is involved in directly activating hut transcription while de-repressing hut translation via the CbrAB-CrcYZ-Crc/Hfq regulatory cascade. Together, our data reveal that the local transcription factor HutC plays a crucial role in governing NtrBC to maintain carbon/nitrogen homeostasis through the complex interactions between two TCSs (NtrBC and CbrAB) at the hut promoter.

Transcriptomic Identification of a Unique Set of Nodule-Specific Cysteine-Rich Peptides Expressed in the Nitrogen-Fixing Root Nodule of Astragalus sinicus.

Legumes in the inverted repeat-lacking clade (IRLC) each produce a unique set of nodule-specific cysteine-rich (NCR) peptides, which act in concert to determine the terminal differentiation of nitrogen-fixing bacteroid. IRLC legumes differ greatly in their numbers of NCR and sequence diversity. This raises the significant question how bacteroid differentiation is collectively controlled by the specific NCR repertoire of an IRLC legume. Astragalus sinicus is an IRLC legume that forms indeterminate nodules with its microsymbiont Mesorhizobium huakuii 7653R. Here, we performed transcriptome analysis of root and nodule samples at 3, 7, 14, 28 days postinoculation with M. huakuii 7653R and its isogenic ∆bacA mutant. BacA is a broad-specificity peptide transporter required for the host-derived NCRs to target rhizobial cells. A total of 167 NCRs were identified in the RNA transcripts. Comparative sequence and electrochemical analysis revealed that A. sinicus NCRs (AsNCRs) are dominated by a unique cationic group (termed subgroup C), whose mature portion is relatively long (>60 amino acids) and phylogenetically distinct and possessing six highly conserved cysteine residues. Subsequent functional characterization showed that a 7653R variant harboring AsNCR083 (a representative of subgroup C AsNCR) displayed significant growth inhibition in laboratory media and formed ineffective white nodules on A. sinicus with irregular symbiosomes. Finally, bacterial two-hybrid analysis led to the identification of GroEL1 and GroEL3 as the molecular targets of AsNCR067 and AsNCR076. Together, our data contribute to a systematic understanding of the NCR repertoire associated with the A. sinicus and M. huakuii symbiosis. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Mutations in surface-sensing receptor WspA lock the Wsp signal transduction system into a constitutively active state.

Pseudomonas aeruginosa rugose small-colony variants (RSCVs) are frequently isolated from chronic infections, yet, they are rarely reported in environmental isolates. Here, during the comparative genomic analysis of two P. aeruginosa strains isolated from crude oil, we discovered a spontaneous in-frame deletion, wspAΔ280-307 , which led to hyper-biofilm and RSCV phenotypes. WspA is a homologue of methyl-accepting chemotaxis proteins (MCPs) that senses surfaces to regulate biofilm formation by stimulating cyclic-di-guanosine monophosphate (c-di-GMP) synthesis through the Wsp system. However, the methylation sites of WspA have never been identified. In this study, we identified E280 and E294 of WspA as methylation sites. The wspAΔ280-307 mutation enabled the Wsp system to lock into a constitutively active state that is independent of regulation by methylation. The result is an enhanced production of c-di-GMP. Sequence alignment revealed three conserved repeat sequences within the amino acid residues 280-313 (aa280-313) region of WspA homologues, suggesting that a spontaneous deletion within this DNA encoding region was likely a result of intragenic recombination and that similar mutations might occur in several related bacterial genera. Our results provide a plausible explanation for the selection of RSCVs and a mechanism to confer a competitive advantage for P. aeruginosa in a crude-oil environment.

Investigating the Involvement of Cytoskeletal Proteins MreB and FtsZ in the Origin of Legume-Rhizobial Symbiosis.

Rhizobia are rod-shaped bacteria that form nitrogen-fixing root nodules on leguminous plants; however, they don't carry MreB, a key determinant of rod-like cell shape. Here, we introduced an actin-like mreB homolog from a pseudomonad into Mesorhizobium huakuii 7653R (a microsymbiont of Astragalus sinicus L.) and examined the molecular, cellular, and symbiotic phenotypes of the resultant mutant. Exogenous mreB caused an enlarged cell size and slower growth in laboratory medium. However, the mutant formed small, ineffective nodules on A. sinicus (Nod+ Fix-), and rhizobial cells in the infection zone were unable to differentiate into bacteroids. RNA sequencing analysis also revealed minor effects of mreB on global gene expression in free-living cells but larger effects for cells grown in planta. Differentially expressed nodule-specific genes include cell cycle regulators such as the tubulin-like ftsZ1 and ftsZ2. Unlike the ubiquitous FtsZ1, an FtsZ2 homolog was commonly found in Rhizobium, Sinorhizobium, and Mesorhizobium spp. but not in closely related nonsymbiotic species. Bacterial two-hybrid analysis revealed that MreB interacts with FtsZ1 and FtsZ2, which are targeted by the host-derived nodule-specific cysteine-rich peptides. Significantly, MreB mutation D283A disrupted the protein-protein interactions and restored the aforementioned phenotypic defects caused by MreB in M. huakuii. Together, our data indicate that MreB is detrimental for modern rhizobia and its interaction with FtsZ1 and FtsZ2 causes the symbiotic process to cease at the late stage of bacteroid differentiation. These findings led to a hypothesis that loss of mreB in the common ancestor of members of Rhizobiales and subsequent acquisition of ftsZ2 are critical evolutionary steps leading to legume-rhizobial symbiosis.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Global Regulatory Roles of the Histidine-Responsive Transcriptional Repressor HutC in Pseudomonas fluorescens SBW25.

HutC is known as a transcriptional repressor specific for histidine utilization (hut) genes in Gram-negative bacteria, including Pseudomonas fluorescens SBW25. However, its precise mode of protein-DNA interactions hasn't been examined with purified HutC proteins. Here, we performed electrophoretic mobility shift assay (EMSA) and DNase I footprinting using His6-tagged HutC and biotin-labeled probe of the hut promoter (PhutU). Results revealed a complex pattern of HutC oligomerization, and the specific protein-DNA interaction is disrupted by urocanate, a histidine derivative, in a concentration-dependent manner. Next, we searched for putative HutC-binding sites in the SBW25 genome. This led to the identification of 143 candidate targets with a P value less than 10-4 HutC interaction with eight selected candidate sites was subsequently confirmed by EMSA analysis, including the type IV pilus assembly protein PilZ, phospholipase C (PlcC) for phosphatidylcholine hydrolyzation, and key regulators of cellular nitrogen metabolism (NtrBC and GlnE). Finally, an isogenic hutC deletion mutant was subjected to transcriptome sequencing (RNA-seq) analysis and phenotypic characterization. When bacteria were grown on succinate and histidine, hutC deletion caused upregulation of 794 genes and downregulation of 525 genes at a P value of <0.05 with a fold change cutoff of 2.0. The hutC mutant displayed an enhanced spreading motility and pyoverdine production in laboratory media, in addition to the previously reported growth defect on the surfaces of plants. Together, our data indicate that HutC plays global regulatory roles beyond histidine catabolism through low-affinity binding with operator sites located outside the hut locus.IMPORTANCE HutC in Pseudomonas is a representative member of the GntR/HutC family of transcriptional regulators, which possess a N-terminal winged helix-turn-helix (wHTH) DNA-binding domain and a C-terminal substrate-binding domain. HutC is generally known to repress expression of histidine utilization (hut) genes through binding to the PhutU promoter with urocanate (the first intermediate of the histidine degradation pathway) as the direct inducer. Here, we first describe the detailed molecular interactions between HutC and its PhutU target site in a plant growth-promoting bacterium, P. fluorescens SBW25, and further show that HutC possesses specific DNA-binding activities with many targets in the SBW25 genome. Subsequent RNA-seq analysis and phenotypic assays revealed an unexpected global regulatory role of HutC for successful bacterial colonization in planta.

Plant transcriptome analysis reveals specific molecular interactions between alfalfa and its rhizobial symbionts below the species level.

BACKGROUND: Leguminous plants alter patterns of gene expression in response to symbiotic colonization and infection by their cognate rhizobial bacteria, but the extent of the transcriptomic response has rarely been examined below the species level. Here we describe the identification of 12 rhizobial biotypes of Ensifer meliloti, which form nitrogen-fixing nodules in the roots of alfalfa (Medicago sativa L.), followed by a comparative RNA-seq analysis of four alfalfa cultivars each inoculated with two E. meliloti strains varying in symbiotic performance and phylogenetic relatedness. RESULTS: Rhizobial biotypes were identified on the basis of their symbiotic performance, particularly shoot dry weight. Differentially expressed genes (DEGs) and metabolic pathways were determined by comparing the RNA-seq data with that of the uninoculated control plant. Significant differences were found between DEGs generated in each cultivar with the inoculation of two rhizobial strains in comparison (P < 0.01). A total of 8111 genes was differentially expressed, representing ~ 17.1% of the M. sativa genome. The proportion of DEGs ranges from 0.5 to 12.2% for each alfalfa cultivar. Interestingly, genes with predicted roles in flavonoid biosynthesis and plant-pathogen interaction (NBS-LRR) were identified as the most significant DEGs. Other DEGs include Medsa002106 and genes encoding nodulins and NCR peptides whose expression is specifically induced during the development of nitrogen-fixing nodules. More importantly, strong significant positive correlations were observed between plant transcriptomes (DEGs and KEGG pathways) and phylogenetic distances between the two rhizobial inoculants. CONCLUSIONS: Alfalfa expresses significantly distinct sets of genes in response to infection by different rhizobial strains at the below-species levels (i.e. biotype or strain). Candidate genes underlying the specific interactions include Medsa002106 and those encoding nodulins and NCR peptides and proteins in the NBS-LRR family.

NfiR, a New Regulatory Noncoding RNA (ncRNA), Is Required in Concert with the NfiS ncRNA for Optimal Expression of Nitrogenase Genes in Pseudomonas stutzeri A1501.

Expression of nitrogenase genes (nifHDK) is strictly regulated at both transcriptional and posttranscriptional levels. Efficient nitrogenase activity requires maintaining sufficient levels of nif mRNAs, yet the underlying mechanism is not fully understood due to its complexity. We have previously shown that a novel regulatory noncoding RNA (ncRNA), NfiS, optimizes nitrogen fixation through targeting nifK mRNA in Pseudomonas stutzeri A1501. Here, we report the identification and characterization of a second ncRNA inducible under nitrogen fixation conditions (nitrogen-free and microaerobic conditions), termed NfiR (for nitrogen fixation condition-inducible ncRNA), the expression of which is dependent on two global regulators, NtrC and Hfq. Comparative phenotypic and proteomic analyses of an nfiR mutant identify a role of NfiR in regulating the expression of nitrogenase genes. Further microscale thermophoresis and genetic complementation showed that an 11-nucleotide (nt) sequence in the stem-loop structure of NfiR (nucleotides 12 to 22) pairs with its counterpart in the coding region of nifD mRNA (nucleotides 1194 to 1207) by eight nucleotides. Significantly, deletion of nfiR caused a 60% reduction of nitrogenase activity, and the half-life of nifD mRNA was reduced from 20 min for the wild type to 15 min for the ΔnfiR mutant. With regard to nitrogenase activity and stability of the nifD and nifK transcripts, phenotypes were more severe for the double deletion mutant lacking nfiR and nfiS, suggesting that NfiR, in concert with NfiS, optimizes nitrogenase production at the posttranscriptional level.IMPORTANCE Biological nitrogen fixation is an energy-expensive process requiring the hydrolysis of 16 ATPs. Consequently, the expression of nif genes is highly regulated at both transcriptional and posttranscriptional levels through complex regulatory networks. Global regulation involves a number of regulatory proteins, such as the nif-specific activator NifA and the global nitrogen regulator NtrC, as well as various regulatory ncRNAs. We show that the two P. stutzeri ncRNAs, namely NfiS and NfiR (for nitrogen fixation condition-inducible ncRNA), optimize nitrogen fixation and environmental stress responses. NfiS and NfiR respond differently to various environmental signals and differ in their secondary structures. In addition, the two ncRNAs target the mRNAs of nifK and nifD, respectively. Such ncRNA-based posttranscriptional regulation of nitrogenase expression might be an evolved survival strategy, particularly in nitrogen-limiting environments. This study not only highlights the significant roles of regulatory ncRNAs in the coordination and fine tuning of various physiological processes but also provides a new paradigm for posttranscriptional regulation in nitrogen-fixing bacteria.

Unravelling the complexity and redundancy of carbon catabolic repression in Pseudomonas fluorescens SBW25.

The two-component system CbrAB is the principal regulator for cellular metabolic balance in Pseudomonas fluorescens SBW25 and is necessary for growth on many substrates including xylose. To understand the regulatory linkage between CbrAB and genes for xylose utilization (xut), we performed transposon mutagenesis of ΔcbrB to select for Xut+ suppressors. This led to identification of crc and hfq. Subsequent genetic and biochemical analysis showed that Crc and Hfq are key mediators of succinate-provoked carbon catabolite repression (CCR). Specifically, Crc/Hfq sequentially bind to mRNAs of both the transcriptional activator and structural genes involved in xylose catabolism. However, in the absence of succinate, repression is relieved through competitive binding by two ncRNAs, CrcY and CrcZ, whose expression is activated by CbrAB. These findings provoke a model for CCR in which it is assumed that crc and hfq are functionally complementary, whereas crcY and crcZ are genetically redundant. Inactivation of either crcY or crcZ produced no effects on bacterial fitness in laboratory media, however, results of mathematical modelling predict that the co-existence of crcY and crcZ requires separate functional identity. Finally, we provide empirical evidence that CCR is advantageous in nutrient-complex environments where preferred carbon sources are present at high concentrations but fluctuate in their availability.

Molecular mechanisms of xylose utilization by Pseudomonas fluorescens: overlapping genetic responses to xylose, xylulose, ribose and mannitol.

Bacterial degradation of xylose is sequentially mediated by two enzymes - an isomerase (XutA) and a xylulokinase (XutB) - with xylulose as an intermediate. Pseudomonas fluorescens SBW25, though capable of growth on xylose as a sole carbon source, encodes only one degradative enzyme XutA at the xylose utilization (xut) locus. Here, using site-directed mutagenesis and transcriptional assays, we have identified two functional xylulokinase-encoding genes (xutB1 and xutB2) and further show that expression of xutB1 is specifically induced by xylose. Surprisingly, xylose-induced xutB1 expression is mediated by the mannitol-responsive regulator MtlR, using xylulose rather than xylose as the direct inducer. In contrast, expression of the xutA operon is regulated by XutR - a transcriptional activator of the AraC family - in a xylose-, xylulose- and ribose-dependent manner. Detailed genetic and biochemical analyses of XutR, including DNase I footprinting assays, suggest an unconventional model of XutR regulation that does not involve DNA-looping, a mechanism typically found for AraC-type regulators from enteric bacteria. XutR functions as a dimer and recognizes two inverted repeat sequences, but binding to one half site is weak thus requiring an inducer molecule such as xylose for activation.

Role of the Transporter-Like Sensor Kinase CbrA in Histidine Uptake and Signal Transduction.

UNLABELLED: CbrA is an atypical sensor kinase found in Pseudomonas. The autokinase domain is connected to a putative transporter of the sodium/solute symporter family (SSSF). CbrA functions together with its cognate response regulator, CbrB, and plays an important role in nutrient acquisition, including regulation of hut genes for the utilization of histidine and its derivative, urocanate. Here we report on the findings of a genetic and biochemical analysis of CbrA with a focus on the function of the putative transporter domain. The work was initiated with mutagenesis of histidine uptake-proficient strains to identify histidine-specific transport genes located outside the hut operon. Genes encoding transporters were not identified, but mutations were repeatedly found in cbrA. This, coupled with the findings of [(3)H]histidine transport assays and further mutagenesis, implicated CbrA in histidine uptake. In addition, mutations in different regions of the SSSF domain abolished signal transduction. Site-specific mutations were made at four conserved residues: W55 and G172 (SSSF domain), H766 (H box), and N876 (N box). The mutations W55G, G172H, and N876G compromised histidine transport but had minimal effects on signal transduction. The H766G mutation abolished both transport and signal transduction, but the capacity to transport histidine was restored upon complementation with a transport-defective allele of CbrA, most likely due to interdomain interactions. Our combined data implicate the SSSF domain of CbrA in histidine transport and suggest that transport is coupled to signal transduction. IMPORTANCE: Nutrient acquisition in bacteria typically involves membrane-bound sensors that, via cognate response regulators, determine the activity of specific transporters. However, nutrient perception and uptake are often coupled processes. Thus, from a physiological perspective, it would make sense for systems that couple the process of signaling and transport within a single protein and where transport is itself the stimulus that precipitates signal transduction to have evolved. The CbrA regulator in Pseudomonas represents a unique type of sensor kinase whose autokinase domain is connected to a transporter domain. We present genetic and biochemical evidence that suggests that CbrA plays a dual role in histidine uptake and sensing and that transport is dependent on signal transduction.

Urocanate as a potential signaling molecule for bacterial recognition of eukaryotic hosts.

Host recognition is the crucial first step in infectious disease pathogenesis. Recognition allows pathogenic bacteria to identify suitable niches and deploy appropriate phenotypes for successful colonization and immune evasion. However, the mechanisms underlying host recognition remain largely unknown. Mounting evidence suggests that urocanate-an intermediate of the histidine degradation pathway-accumulates in tissues, such as skin, and acts as a molecule that promotes bacterial infection via molecular interaction with the bacterial regulatory protein HutC. In Gram-negative bacteria, HutC has long been known as a transcriptional repressor of hut genes for the utilization of histidine (and urocanate) as sources of carbon and nitrogen. Recent work on the opportunistic human pathogen Pseudomonas aeruginosa and zoonotic pathogen Brucella abortus shows that urocanate, in conjunction with HutC, plays a significant role in the global control of cellular metabolism, cell motility, and expression of virulence factors. We suggest that in addition to being a valuable source of carbon and nitrogen, urocanate may be central to the elicitation of bacterial pathogenesis.

The biosurfactant viscosin produced by Pseudomonas fluorescens SBW25 aids spreading motility and plant growth promotion.

Food security depends on enhancing production and reducing loss to pests and pathogens. A promising alternative to agrochemicals is the use of plant growth-promoting rhizobacteria (PGPR), which are commonly associated with many, if not all, plant species. However, exploiting the benefits of PGPRs requires knowledge of bacterial function and an in-depth understanding of plant-bacteria associations. Motility is important for colonization efficiency and microbial fitness in the plant environment, but the mechanisms employed by bacteria on and around plants are not well understood. We describe and investigate an atypical mode of motility in Pseudomonas fluorescens SBW25 that was revealed only after flagellum production was eliminated by deletion of the master regulator fleQ. Our results suggest that this 'spidery spreading' is a type of surface motility. Transposon mutagenesis of SBW25ΔfleQ (SBW25Q) produced mutants, defective in viscosin production, and surface spreading was also abolished. Genetic analysis indicated growth-dependency, production of viscosin, and several potential regulatory and secretory systems involved in the spidery spreading phenotype. Moreover, viscosin both increases efficiency of surface spreading over the plant root and protects germinating seedlings in soil infected with the plant pathogen Pythium. Thus, viscosin could be a useful target for biotechnological development of plant growth promotion agents.

Regulatory roles of the second messenger c-di-GMP in beneficial plant-bacteria interactions.

The rhizosphere system of plants hosts a diverse consortium of bacteria that confer beneficial effects on plant, such as plant growth-promoting rhizobacteria (PGPR), biocontrol agents with disease-suppression activities, and symbiotic nitrogen fixing bacteria with the formation of root nodule. Efficient colonization in planta is of fundamental importance for promoting of these beneficial activities. However, the process of root colonization is complex, consisting of multiple stages, including chemotaxis, adhesion, aggregation, and biofilm formation. The secondary messenger, c-di-GMP (cyclic bis-(3'-5') dimeric guanosine monophosphate), plays a key regulatory role in a variety of physiological processes. This paper reviews recent progress on the actions of c-di-GMP in plant beneficial bacteria, with a specific focus on its role in chemotaxis, biofilm formation, and nodulation.

Evaluation of the probiotic potential of yeast isolated from kombucha in New Zealand.

The current study investigated the in vitro probiotic potential of yeast isolated from kombucha, a tea beverage fermented with a symbiotic culture of acetic acid bacteria and yeast. A total of 62 yeast strains were previously isolated from four different commercial kombucha samples sold in New Zealand. Fifteen representative isolates belonging to eight different species were evaluated for their growth under different conditions (temperature, low pH, concentrations of bile salts, and NaCl). Cell surface characteristics, functional and enzymatic activities of the selected strains were also studied in triplicate experiments. Results showed that six strains (Dekkera bruxellensis LBY1, Sachizosaccharomyces pombe LBY5, Hanseniaspora valbyensis DOY1, Brettanomyces anomalus DOY8, Pichia kudraivzevii GBY1, and Saccharomyces cerevisiae GBY2) were able to grow under low-acid conditions (at pH 2 and pH 3) and in the presence of bile salts. This suggests their potential to survive passage through the human gut. All 15 strains exhibited negative enzymatic activity reactions (haemolytic, gelatinase, phospholipase, and protease activities), and thus, they can be considered safe to consume. Notably, two of the fifteen strains (Pichia kudraivzevii GBY1 and Saccharomyces cerevisiae GBY2) exhibited desirable cell surface hydrophobicity (64.60-83.87%), auto-aggregation (>98%), co-aggregation, resistance to eight tested antibiotics (ampicillin, chloramphenicol, colistin sulphate, kanamycin, nalidixic acid, nitrofurantoin, streptomycin, and tetracycline), and high levels of antioxidant activities (>90%). Together, our data reveal the probiotic activities of two yeast strains GBY1 and GBY2 and their potential application in functional food production.

Assessing the relative effects of geographic location and soil type on microbial communities associated with straw decomposition.

Decomposition of plant residues is largely mediated by soil-dwelling microorganisms whose activities are influenced by both climate conditions and properties of the soil. However, a comprehensive understanding of their relative importance remains elusive, mainly because traditional methods, such as soil incubation and environmental surveys, have a limited ability to differentiate between the combined effects of climate and soil. Here, we performed a large-scale reciprocal soil transplantation experiment, whereby microbial communities associated with straw decomposition were examined in three initially identical soils placed in parallel in three climate regions of China (red soil, Chao soil, and black soil, located in midsubtropical, warm-temperate, and cold-temperate zones). Maize straws buried in mesh bags were sampled at 0.5, 1, and 2 years after the burial and subjected to chemical, physical, and microbiological analyses, e.g., phospholipid fatty acid analysis for microbial abundance, community-level physiological profiling, and 16S rRNA gene denaturing gradient gel electrophoresis, respectively, for functional and phylogenic diversity. Results of aggregated boosted tree analysis show that location rather soil is the primary determining factor for the rate of straw decomposition and structures of the associated microbial communities. Principal component analysis indicates that the straw communities are primarily grouped by location at any of the three time points. In contrast, microbial communities in bulk soil remained closely related to one another for each soil. Together, our data suggest that climate (specifically, geographic location) has stronger effects than soil on straw decomposition; moreover, the successive process of microbial communities in soils is slower than those in straw residues in response to climate changes.

Immigration history controls diversification in experimental adaptive radiation.

Diversity in biological communities is a historical product of immigration, diversification and extinction, but the combined effect of these processes is poorly understood. Here we show that the order and timing of immigration controls the extent of diversification. When an ancestral bacterial genotype was introduced into a spatially structured habitat, it rapidly diversified into multiple niche-specialist types. However, diversification was suppressed when a niche-specialist type was introduced before, or shortly after, introduction of the ancestral genotype. In contrast, little suppression occurred when the same niche specialist was introduced relatively late. The negative impact of early arriving immigrants was attributable to the historically sensitive outcome of interactions involving neutral competition and indirect facilitation. Ultimately, the entire boom-and-bust dynamics of adaptive radiation were altered. These results demonstrate that immigration and diversification are tightly linked processes, with small differences in immigration history greatly affecting the evolutionary emergence of diversity.

Bioaugmentation of woodchip bioreactors by Pseudomonas nicosulfuronedens D1-1 with functional species enrichment.

A novel heterotrophic nitrification and aerobic denitrification (HN-AD) bacterium D1-1 was identified as Pseudomonas nicosulfuronedens D1-1. Strain D1-1 removed 97.24%, 97.25%, and 77.12% of 100 mg/L NH4+-N, NO3--N, and NO2--N, with corresponding maximum removal rates of 7.42, 8.69, and 7.15 mg·L-1·h-1, respectively. Strain D1-1 bioaugmentation enhanced woodchip bioreactor performance with an average NO3--N removal efficiency of 93.8%. Bioaugmentation enriched N cyclers along with increased bacterial diversity and predicted genes for denitrification, DNRA (dissimilatory nitrate reduction to ammonium), and ammonium oxidation. It also reduced local selection and network modularity from 4.336 to 0.934, resulting in predicted nitrogen (N) cycling genes shared by more modules. These observations suggested that bioaugmentation could enhance the functional redundancy to stabilize the NO3--N removal performance. This study provides insights into the potential applications of HN-AD bacteria in bioremediation or other environmental engineering fields, relying on their ability to shape bacterial communities.

Microbiological and Physico-Chemical Characteristics of Black Tea Kombucha Fermented with a New Zealand Starter Culture.

Kombucha is a popular sparkling sugared tea, fermented by a symbiotic culture of acetic acid bacteria (AAB) and yeast. The demand for kombucha continues to increase worldwide, mainly due to its perceived health benefits and appealing sensory properties. This study isolated and characterised the dominant AAB and yeast from a starter culture and kombucha broth after 0, 1, 3, 5, 7, 9, 11, and 14 days of fermentation at ambient temperature (22 °C). Yeast and AAB were isolated from the Kombucha samples using glucose yeast extract mannitol ethanol acetic acid (GYMEA) and yeast extract glucose chloramphenicol (YGC) media, respectively. The phenotypic and taxonomic identification of AAB and yeast were determined by morphological and biochemical characterisation, followed by a sequence analysis of the ribosomal RNA gene (16S rRNA for AAB and ITS for yeast). The changes in the microbial composition were associated with variations in the physico-chemical characteristics of kombucha tea, such as pH, titratable acidity, and total soluble solids (TSS). During fermentation, the acidity increased and the TSS decreased. The yield, moisture content, and water activity of the cellulosic pellicles which had developed at the end of fermentation were attributed to the presence of AAB. The dominant AAB species in the cellulosic pellicles and kombucha broth were identified as Komagataeibacter rhaeticus. The yeast isolates belonged to Debaryomyces prosopidis and Zygosaccharomyces lentus.

Variation in transport explains polymorphism of histidine and urocanate utilization in a natural Pseudomonas population.

Phenotypic variation is a fundamental requirement for evolution by natural selection. While evidence of phenotypic variation in natural populations abounds, its genetic basis is rarely understood. Here we report variation in the ability of plant-colonizing Pseudomonas to utilize histidine, and its derivative, urocanate, as sole sources of carbon and nitrogen. From a population of 164 phyllosphere-colonizing Pseudomonas strains, 77% were able to utilize both histidine and urocanate (His(+) , Uro(+) ) as growth substrates, whereas the remainder could utilize histidine, but not urocanate (His(+) , Uro(-) ), or vice versa (His(-) , Uro(+) ). An in silico analysis of the hut locus, which determines capacity to utilize both histidine and urocanate, from genome-sequenced Pseudomonas strains, showed significant variation in the number of putative transporters. To identify transporter genes specific for histidine and urocanate, we focused on a single genotype of Pseudomonas fluorescens, strain SBW25, which is capable of utilizing both substrates. Site-directed mutagenesis, combined with [(3) H]histidine transport assays, shows that hutT(u) encodes a urocanate-specific transporter; hutT(h) encodes the major high-affinity histidine transporter; and hutXWV encodes an ABC-type transporter that plays a minor role in histidine uptake. Introduction of cloned copies of hutT(h) and hutT(u) from SBW25 into strains incapable of utilizing either histidine, or urocanate, complemented the defect, demonstrating a lack of functional transporters in these strains. Taken together our data show that variation in transport systems, and not in metabolic genes, explains a naturally occurring phenotypic polymorphism.