Interestingly, H. seropedicae SmR1 genes related to PHB metabolism appeared to affect fitness by enhancing or decreasing root colonization as shown in Fig. 4. Among the nine selected genes where mutations impaired colonization by H. seropedicae , six mutants—specifically the opuBC, typA, ampD, purD, exbD, and exbB mutants—showed a significant reduction in root colonization either when inoculated separately or coinoculated with the wild type .Applying the transposon mutagenesis sequencing approach to both A. olearius DQS4T and H. seropedicae SmR1 revealed many genes required for these bacteria to competitively colonize Setaria viridis roots. Our experiments identified 89 and 130 genes where mutations significantly affected the ability of A. olearius or H. seropedicae, respectively, to colonize S. viridis roots, including some genes previously reported to play a role in the plant-microbe associations. This result alone argues that root colonization is not a simple process but one that involves a variety of bacterial functions. General gene classes include those involved in cell wall biosynthesis, motility, chemotaxis and defense, and amino acid metabolism . Colonization assays with the 15 candidate genes selected for further confirmation by insertional mutagenesis showed a strong correlation with the results obtained by TnSeq, giving confidence that most if not all of the genes identified are likely important for root colonization by these PGPB strains. Genes where mutations benefited root colonization can be assumed to normally play a role in suppressing colonization in wild-type cells. Among such genes in A. olearius DQS4T is a homolog of the BH72 exaA5 gene ,large growing pots predicted to encode a pyrroloquinoline quinone-dependent alcohol dehydrogenase involved in methanol oxidation.
Consistent with these findings, previous reports showed that mutation of ADH genes inhibited competitive colonization of rice roots by A. olearius BH72 . Similarly, Methylobacterium spp. mutants defective in methanol oxidation were less competitive for colonization of Medicago truncatula roots when coinoculated with wild type cells . These data suggest that methanol metabolism is important for bacterial growth on the root surface and, perhaps more importantly, colonization is a very dynamic and likely heavily influenced by the overall microbial community. We observed genes that increased fitness scores clustered within an operon presumably involved in iron uptake in Azoarcus. These genes are predicted to encode an outer membrane, ferric coprogen protein FhuE , and a TonB-dependent siderophore receptor , both of which were previously implicated in the ability of this bacterium to colonize roots . Bacterial iron uptake is complex, perhaps involving multiple bacterial processes, and can also be coopted by plant-encoded mechanisms. Mutation of these genes increased fitness values conveying a phenotypic advantage during root colonization, although less competitive than the wild type. Given the general role that iron availability plays in the ability of microorganisms to thrive and compete in virtually any environment, it is not surprising that iron uptake is also a crucial function for root colonization . Recently, analysis of iron content in maize treated with the PGPB A. brasilense revealed a significant increase in total iron accumulation in seeds and higher yield , suggesting that PGPB can contribute to the iron metabolism of the host plant. Previous studies demonstrated an important role for bacterial genes involved in motility for both endophytic and rhizosphere colonization of host roots.
Many genes involved in cell motility were among the common COG categories that appear to provide a fitness advantage for root colonization by SmR1, specifically mutations in genes related to flagellum assembly. We showed previously that an H. seropedicae mutant in the flagellar regulatory gene fliA was unable to endophytically colonize S. viridis roots, although this mutation did not affect rhizosphere colonization . fliA encodes the sigma factor s28 RNA polymerase that mediates the transcription of genes involved in motility and flagellar synthesis .However, bacterial flagella can be recognized by specific receptors in plant cell membranes and activate a cascade of immune responses controlling bacterial infection . Transcriptome analysis of SmR1 attached to wheat roots showed that the flagellar gene cluster was down regulated, suggesting that the bacteria might switch to a twitching type of motility mediated by type IV pili . Under certain environmental stresses or nutritional conditions, bacteria can use different sources of energy for survival, including mobilization of polymers such as polyhydroxyalkanoates . PHB is the PHA produced by bacteria. The PHB granules act as carbon storage that can be mobilized under different conditions. We found that disruption of PHA depolymerase, encoded by the phaZ1 gene, enhanced bacterial colonization when inoculated individually or in competition with the wild type. According to Silveira Alves et al. , plant biomass was significantly reduced in S. viridis colonized by DphaZ1 mutant despite colonizing roots to the same level as the wild-type strain. In contrast, we identified PhaP1 encoding a phasin involved in the PHB production , where deletion of DphaP1 affected fitness negatively, reducing root colonization. Corroborating our findings, an increase in gene expression of the phasin genes was reported during wheat root colonization . Many genes involved in peptidoglycan and lipopolysaccharide biosynthesis were predicted to impair bacterial colonization of plant roots.
For instance, mutation of LPS biosynthetic genes or the addition of exogenous N-acetylglucosamine was previously shown to impair H. seropedicae-maize root association . In studies of rice roots colonized by Azoarcus BH72, mutation of an endoglucanase reduced root colonization, suggesting its importance for successful host cell invasion . We demonstrated that a mutation in a transcriptional regulator involved in aromatic compound degradation completely impaired root colonization during single inoculation. AphS was predicted to regulate genes important for phenol degradation in A. olearius BH72 . Only two genes, cheY involved in chemotaxis and ampD involved in peptidoglycan cell wall recycling ,blueberry planter affected colonization of both strains. Our data suggest that bacterial chemotaxis provides a competitive advantage to wild-type cells during colonization of the plant root tissue. This system is well characterized in several motile bacterial species, such as E. coli and beneficial bacteria such as Azospirillum brasilense, S. meliloti, and others . In summary, our data indicate that, rather than a single or small subset of crucial functions, each strain uses differing functions for colonization, reflecting the unique characteristics of each bacterium. Given that our study was focused on bacterial root colonization, mutations related to soil survival were not considered. However, for Azoarcus a deeper investigation of mutations that affected survival in soil could be useful in explaining the different lifestyle and adaptions of each bacterium to their environment, especially considering that Azoarcus sp. BH72, closely related to A. olearius DQS4T , is a strict endophyte and has not been reported to survive without a host . In summary, similar to most plant-microbe interactions, PGPB-plant interactions are complex and reflect the ability of the plant host and bacterial symbiont to profoundly influence the metabolism of the other. The fact that PGPB have broad host ranges and can enhance crop yield under field conditions has contributed to a continuing interest in using PGPB inoculants in agriculture. This study provides insights to better understand those gene functions involved in PGPB-host interaction and hopefully will contribute to the further development of PGPB inoculants for an efficient, sustainable agriculture.This document provides best practice guidance and energy efficiency recommendations for the design, construction, and operation of high-performance office buildings in India. Through a discussion of learnings from exemplary projects and inputs from experts, it provides recommendations that can potentially help achieve enhanced working environments, economic construction/faster payback, reduced operating costs, and reduced greenhouse gas emissions. It also provides ambitious energy performance benchmarks, both as adopted targets during building modeling and during measurement and verification . These benchmarks have been derived from a set of representative best-in-class office buildings in India.
The best practices strategies presented in this guide would ideally help in delivering high-performance in terms of a triad—of energy efficiency, cost efficiency, and occupant comfort and well-being. These best practices strategies and metrics should be normalized—that is, corrected to account for building characteristics, diversity of operations, weather, and materials and construction methods. Best practices should start by using early design principles at the whole building level. Optimal energy efficiency can be achieved through an integrated design process , with stakeholder buy-in from the beginning at the conceptual design phase. Early in the project, the focus of the stakeholder group should be on maximizing energy efficiency of the building as a whole, and not just on the efficiency of an individual building component or system. Through multi-disciplinary interactions, the design team should explore synergies between systems such as mutually resonating strategies; or sweet spots between inharmonious strategies. Buildings are the most energy efficient when designers and operators ensure that systems throughout the building are both efficient themselves, and work efficiently together. Systems integration and operational monitoring at the whole building level can help push the envelope for building energy efficiency and performance to unprecedented levels. Whole-building systems integration throughout the building’s design, construction, and operation can assure high performance, both in terms of ensures the energy efficiency and comfort/service levels. A Life cycle Performance Assurance Framework emphasizes the critical integration between the buildings’ physical systems and the building information technologies. The building physical systems include envelope, HVAC, plugs, lighting and comfort technology systems. Whereas, building information technologies provide information on the design and functioning of the building physical systems. This can be done- first, by performing building energy simulation and modeling at the design phase one can estimate the building’s energy performance and code compliance; second, by integrating controls and sensors for communications, one can track real-time performance at the building phase, relative to the original design intent; and third, by conducting monitoring-based commissioning and bench marking during operations, one can ascertain building performance compared to peers and provide feedback loops. The next step should be asesssing best practices at the systems and components level along four intersecting building physical systems- Mechanical Systems for Heating, Ventilation and Air Conditioning , Plug Loads, Lighting and Envelope/Passive systems. The qualitative best practices described in this guide offer opportunities for building designers, owners, and operators to improve energy efficiency in commercial office buildings. Although the practices are presented individually, they should not be thought of as an “a la carte” menu of options. Rather, building systems must be integrated to realize the maximum energy and cost benefits. Also, designers and engineers, and developers and tenants need to work together to capitalize on the synergies between systems. Last but not the least, this guide provides tangible quantitative best performance metrics, ready to be adopted by buildings in India. These metrics are concrete targets for stakeholder groups to work together and enable, by providing localized and customized solutions for each building, class, and occupants. Having targets early on in the design process also translates to more-efficient design lead times. The potential benefits of adopting these metrics include efficient operations, first-cost and life cycle cost efficiencies, and occupant comfort and well-being. The best practice strategies, if used thoughtfully provide an approach towards enabling office buildings that would deliver throughout their entire life cycle, a flexible optimization of energy consumption, productivity, safety, comfort and healthfulness. The adoption of the qualitative and quantitative goals, would provide an impetus to scale up and market transformation toward energy-efficient processes, resources, and products- in addition to generating positive outcomes on global warming and societal benefits. This guide’s primary goal is to provide meaningful information on framing building systems performance and guiding important decisions from conceptual design of a building to its operations and maintenance . It focuses on resource and energy-efficient solutions for high performance conditioned offices , with spillover benefits to a diversity of building types, such as retail, hospitality, hospitals, and multi-storied housing. The secondary goal is to initiate a useful, structured repository of design wisdom that can be continually refined and updated over the years in order to time-test the effectiveness of its recommendations and document them. The authors look forward to integrating more ‘Data Points’ and information from buildings in India especially as the bar for best-in-class high-performance buildings is being continually raised. We intend to refine the best practices and metrics, in newer and revised versions of this guide.