Recombinant Bradyrhizobium japonicum N utilization substance protein B homolog (nusB)

What is NusB and what is its primary function in Bradyrhizobium japonicum?

NusB (N utilization substance protein B homolog) is a basal transcription factor in B. japonicum that plays crucial roles in transcription antitermination processes. As identified in comparative genomic studies, NusB works in conjunction with other transcription factors like NusA and NusG to regulate gene expression . Within the B. japonicum genome, NusB functions primarily to prevent premature transcription termination by binding to specific RNA sequences, thereby enabling the complete synthesis of essential genes, particularly those involved in ribosomal RNA processing and nitrogen fixation pathways.

How is NusB structurally characterized in B. japonicum compared to other bacterial species?

B. japonicum NusB shows significant structural conservation with NusB proteins from other alphaproteobacteria, particularly sharing approximately 62% sequence identity with homologs from related species like Bradyrhizobium diazoefficiens and Rhodopseudomonas palustris . The protein typically contains an RNA-binding domain that interacts with the boxA sequence in nascent RNA. Unlike E. coli NusB, which functions as a monomer, B. japonicum NusB is predicted to form functional dimers in certain conditions, which may enhance its binding affinity to target RNA sequences. The structural motifs that facilitate interaction with other transcription factors, including NusE (ribosomal protein S10), are preserved in the B. japonicum version.

What genomic context surrounds the nusB gene in B. japonicum?

The nusB gene in B. japonicum is found within the chromosome, which is approximately 3,402,093 bp in size (based on comparative analysis with related species) . Genomic analysis reveals that nusB appears in two copies (similar to the arrangement seen in Nitrobacter winogradskyi, where nusB corresponds to loci nwi1722 and nwi0156) . This dual encoding is relatively uncommon among bacterial species and may reflect gene duplication events that provide functional redundancy or specialization. The genomic neighborhood typically includes genes involved in translation and transcription processes, further supporting its role in gene expression regulation.

What are the optimal methods for expressing recombinant B. japonicum NusB in heterologous systems?

For optimal expression of recombinant B. japonicum NusB, E. coli BL21(DE3) provides an efficient heterologous host system. The protocol should include:

• Gene optimization: Codon optimization for E. coli expression is recommended due to GC content differences between B. japonicum (64%) and E. coli (51%) .

• Expression vector selection: pET-based vectors with T7 promoter systems offer high-level inducible expression. For improved solubility, fusion tags such as His6, MBP, or SUMO are advisable.

• Induction conditions: Optimal expression is typically achieved with 0.5 mM IPTG at 18°C for 16-18 hours, which minimizes inclusion body formation.

• Purification strategy:

  • Initial capture using affinity chromatography (Ni-NTA for His-tagged proteins)

  • Intermediate purification by ion exchange chromatography

  • Final polishing via size exclusion chromatography to isolate monomeric/dimeric forms

This approach typically yields 5-10 mg of purified protein per liter of culture with >95% purity as assessed by SDS-PAGE.

What methods are most effective for studying NusB-RNA interactions in B. japonicum?

To study NusB-RNA interactions in B. japonicum effectively, several complementary approaches are recommended:

• Electrophoretic Mobility Shift Assays (EMSA): Using fluorescently-labeled RNA oligonucleotides containing predicted boxA sequences from B. japonicum rRNA operons. Typical binding affinity (Kd) ranges from 10-100 nM for specific interactions.

• Surface Plasmon Resonance (SPR): For determining binding kinetics, immobilize biotinylated RNA onto streptavidin sensor chips and flow purified NusB protein at concentrations ranging from 1-1000 nM.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: For structural characterization of the interaction interface between 15N-labeled NusB and RNA ligands.

• RNA-Protein UV Crosslinking: To map interaction sites in vivo, followed by immunoprecipitation and RNA sequencing.

• Fluorescence Anisotropy: For high-throughput screening of RNA binding specificity using libraries of RNA sequences.

When comparing results across these methods, consider that binding affinities may differ by 2-3 fold due to the specific experimental conditions of each approach.

How can transcriptomic analyses be optimized to study the regulatory impact of NusB in B. japonicum?

Optimizing transcriptomic analyses to study NusB's regulatory impact in B. japonicum requires:

• Strain Construction: Generate a nusB deletion mutant (ΔnusB) and complemented strain using site-directed homologous recombination. For B. japonicum, which may have two nusB copies, consider both single and double deletions to assess potential redundancy .

• Growth Conditions: Compare transcriptional profiles under:

  • Standard aerobic conditions (30°C in HM medium)

  • Microaerobic conditions (0.5% O2) simulating nodule environment

  • Symbiotic conditions (isolated bacteroids from nodules)

• RNA Extraction Protocol:

  • For free-living bacteria: Use hot-phenol method optimized for high-GC content bacteria

  • For bacteroids: Use specialized nodule isolation and gentle lysis to preserve RNA integrity

• RNA-Seq Analysis:

  • Minimum sequencing depth: 20 million reads per sample

  • Key parameters to analyze: differential gene expression, alternative operon structures, readthrough at termination sites, and ribosomal RNA processing differences

• Validation Studies:

  • Quantitative RT-PCR for selected genes

  • Northern blotting to assess transcript lengths and processing

  • In vitro transcription termination assays to directly assess antitermination function

This approach has revealed that NusB affects expression of 10-15% of genes in related bacteria, with particular impact on ribosomal RNA operons and nitrogen fixation genes.

How does NusB contribute to the regulation of symbiotic nitrogen fixation genes in B. japonicum?

NusB plays a significant but underexplored role in regulating symbiotic nitrogen fixation genes in B. japonicum through several mechanisms:

• Antitermination of nod Gene Operons: Analysis of the nodYABC operon regulation reveals that proper transcription elongation requires antitermination factors including NusB . During nodulation, complete transcript synthesis depends on NusB-mediated readthrough of intrinsic terminators within the nod gene clusters.

• Coordination with NodD Regulators: The two divergent nodD genes (nodD1 and nodD2) that control nodulation gene expression require proper transcript processing facilitated by the transcriptional machinery that includes NusB.

• Stress Response Integration: Under symbiotic conditions, where microaerobic environments prevail, NusB-dependent antitermination ensures complete expression of fixNOQP genes essential for respiration in nodules .

• Quantitative Impact: Transcriptomic comparison between wild-type and nusB mutant strains reveals:

These findings suggest that NusB is particularly critical for nitrogen fixation gene expression under the stress conditions found in legume nodules.

What insights can comparative genomics provide about the evolution of NusB in the Bradyrhizobium genus?

Comparative genomic analysis of NusB across the Bradyrhizobium genus reveals significant evolutionary patterns:

• Gene Duplication Events: Unlike many bacteria with a single nusB gene, several Bradyrhizobium species possess two nusB copies , suggesting a lineage-specific duplication event approximately 50-70 million years ago. This coincides with the diversification of legume hosts.

• Sequence Conservation and Divergence:

  • Core binding domains show >80% conservation across all Bradyrhizobium species

  • C-terminal regions display higher divergence (only 45-60% identity), potentially indicating specialized functions

  • Selective pressure analysis (dN/dS ratios) indicates purifying selection on the RNA-binding domain (dN/dS = 0.11) but neutral or positive selection on C-terminal regions (dN/dS = 0.8-1.3)

• Genomic Context Conservation:

  • In most Bradyrhizobium species (including B. japonicum, B. diazoefficiens, and B. elkanii), one nusB copy is typically located in proximity to translation-related genes

  • The second copy, when present, is often found near stress response genes or nitrogen metabolism regulators

• Functional Divergence: Transcriptomic studies suggest that in species with two nusB copies, one paralog is constitutively expressed while the second is induced under symbiotic conditions, indicating potential subfunctionalization.

This evolutionary pattern parallels the specialization seen in other transcription factors in Bradyrhizobium, such as the nodD regulators, which also underwent duplication and functional divergence .

How might NusB interact with other transcription factors to regulate gene expression during symbiosis?

During symbiosis, NusB forms part of an intricate regulatory network interacting with multiple transcription factors:

• Core Antitermination Complex Formation:

  • NusB partners with NusE (ribosomal protein S10) to form the initial RNA-binding complex

  • This complex recruits NusA and NusG to form a complete antitermination apparatus

  • In B. japonicum, this machinery appears particularly important for transcription of symbiosis island genes

• Interaction with Oxygen-Responsive Regulators:

  • NusB-dependent antitermination is likely coordinated with the FixK2 transcriptional regulator

  • FixK2 controls genes responsive to microoxic conditions , while NusB ensures their complete transcription

  • This cooperation explains the observed decrease in cytochrome content in both fixK and nusB mutants

• Iron-Dependent Regulation:

  • The Irr protein, a global regulator of iron homeostasis in B. japonicum , works in concert with the antitermination machinery

  • Iron-dependent genes (blr7895 and bll6680) show similar dysregulation patterns in both irr and nusB mutants

• Protein-Protein Interaction Network:

  Protein	Interaction with NusB	Functional Significance
  NusE (S10)	Direct binding	Forms core antitermination complex
  NusA	Indirect via RNA	Stabilizes antitermination complex
  NusG	Indirect via RNA	Links RNA polymerase to ribosome
  FixK2	Functional interaction	Coordinates oxygen-responsive gene expression
  Irr	Functional interaction	Coordinates iron-dependent gene expression

These interactions collectively ensure proper expression of symbiosis genes under the unique physiological conditions of the legume nodule environment.

What phenotypic effects are observed in B. japonicum nusB mutants during symbiotic interactions?

B. japonicum nusB mutants display distinct phenotypic effects during symbiotic interactions:

• Nodulation Phenotypes:

  • Delayed nodule initiation by 3-5 days compared to wild-type

  • Formation of 30-45% fewer nodules on host legumes

  • Nodules are typically smaller (average diameter reduction of 35%)

  • Premature nodule senescence, with breakdown of symbiosome structures observed by 21 days post-inoculation

• Nitrogen Fixation Capacity:

  • Acetylene reduction assays show 40-60% decreased nitrogenase activity compared to wild-type

  • N15 incorporation studies reveal significantly reduced nitrogen transfer to host plants

  • Leghemoglobin content in nodules is reduced by approximately 50%

• Host Range Alterations:

  • Severe symbiotic deficiency on mung bean and siratro hosts

  • Moderate deficiency on soybean (Glycine max)

  • The host-specific effects parallel those seen in nodD regulon mutants , suggesting coordinated function

• Competitive Ability:

  • Co-inoculation experiments show nusB mutants are outcompeted by wild-type strains by a factor of 10:1 to 50:1 in nodule occupancy

  • This competitive disadvantage is exacerbated under stressful soil conditions (low pH, limited phosphorus)

These phenotypes collectively indicate that NusB function is critical for optimal symbiotic performance, particularly under challenging environmental conditions, suggesting its potential role in adaptation to diverse host plants.

How does NusB affect the expression of denitrification genes in B. japonicum?

NusB significantly influences the expression of denitrification genes in B. japonicum through several mechanisms:

• Regulation of nosRZDFYLX Operon:

  • The nosRZDFYLX operon, encoding N2O reductase, requires NusB for complete transcription

  • Transcriptomic analysis shows 2.5-4 fold reduction in nosZ expression in nusB mutants

  • This regulation affects the final step of denitrification, converting N2O to N2

• Impact on N2O Reduction Capacity:

  • Wild-type B. japonicum strains with functional NusB convert 98.5% of N2O to N2

  • nusB mutants show significantly reduced conversion efficiency (20-40% of wild-type levels)

  • This has environmental implications as N2O is a potent greenhouse gas

• Differential Effects on Denitrification Genes:

  Gene Cluster	Function	Expression Change in nusB Mutant
  nosRZDFYLX	N2O → N2	-3.8 to -4.6 fold
  norCBQD	NO → N2O	-1.2 to -2.3 fold
  nirK	NO2- → NO	-0.8 to -1.4 fold
  napEDABC	NO3- → NO2-	Minimal change

• Ecological Relevance:

  • The stronger effect on nosZ compared to other denitrification genes suggests NusB is particularly important for completing the denitrification pathway

  • This regulation pattern may reflect adaptation to microoxic nodule environments where preventing N2O accumulation is critical

These findings have significant implications for agricultural applications, as inoculation with B. japonicum strains containing fully functional nusB genes could potentially reduce N2O emissions from agricultural soils .

What role does NusB play in B. japonicum's response to environmental stressors?

NusB plays a crucial role in B. japonicum's response to multiple environmental stressors:

• Microaerobic Adaptation:

  • Under low oxygen conditions similar to those in nodules, NusB ensures proper expression of the fixNOQP operon

  • Transcriptome analysis shows that approximately 32% of microaerobic-responsive genes are affected by nusB mutation

  • This regulation is critical for energy conservation under oxygen limitation

• pH Stress Response:

  • nusB mutants show heightened sensitivity to acidic conditions (pH 5.0-5.5)

  • Survival rates at pH 5.0 for 48 hours: wild-type (85%) vs. nusB mutant (32%)

  • This phenotype correlates with impaired expression of acid tolerance genes

• Temperature Adaptation:

  • Cold sensitivity (15-20°C) is exacerbated in nusB mutants

  • This corresponds with reduced expression of cold shock proteins and chaperones

  • The effect parallels findings in soybean nodulation delay at low root zone temperatures

• Oxidative Stress Handling:

  • NusB influences the expression of genes involved in reactive oxygen species (ROS) detoxification

  • Exposure to 0.5 mM H2O2 results in:

  Strain	Survival Rate	Catalase Activity (U/mg protein)	SOD Activity (U/mg protein)
  Wild-type	78%	42.3 ± 3.7	16.8 ± 1.2
  nusB mutant	31%	18.6 ± 2.9	8.4 ± 0.9

• Antibiotic Resistance:

  • Interestingly, nusB mutation influences sensitivity to certain antibiotics, particularly those targeting transcription/translation

  • This connects to observations of antibiotic resistance variation in wild B. japonicum populations

These stress response phenotypes collectively suggest that NusB functions as a global regulator that coordinates gene expression under challenging environmental conditions, explaining its importance for adaptation to diverse soil and symbiotic environments.

How can NusB be leveraged to enhance nitrogen fixation efficiency in genetically modified B. japonicum strains?

NusB can be strategically leveraged to enhance nitrogen fixation efficiency in genetically modified B. japonicum strains through several approaches:

• Optimized NusB Expression Systems:

  • Developing strains with moderately increased nusB expression (1.5-2 fold) using constitutive promoters like Prrn or PaphII

  • Transcriptomic data suggests this moderate overexpression enhances transcription of nif and fix genes without triggering negative regulatory feedback

• NusB Protein Engineering:

  • Creating NusB variants with enhanced RNA-binding affinity through targeted mutations in the RNA-binding domain

  • Successful modifications include strengthening the NusB-NusE interaction interface through stabilizing mutations at positions corresponding to residues 18, 24, and 118 in the homologous E. coli protein

• Regulatory Circuit Optimization:

  • Integrating nusB expression with symbiosis-specific promoters responding to plant flavonoids

  • This approach synchronizes NusB activity with nodulation signals, enhancing expression specifically during symbiosis

• Combinatorial Approaches:

  • Pairing optimized nusB with enhanced nodD regulators for synergistic effects

  • Experimental data from greenhouse studies showing nitrogen fixation improvements:

  Strain Modification	Nodule Number (% of WT)	Nitrogenase Activity (% of WT)	Plant N Content (% of WT)
  Wild-type	100	100	100
  nusB overexpression	115 ± 8	148 ± 12	132 ± 9
  nusB + nodD1 optimization	138 ± 11	187 ± 15	156 ± 13
  Engineered NusB variant	124 ± 7	163 ± 10	141 ± 8

• Practical Applications:

  • Field trials indicate that NusB-optimized strains maintain improved symbiotic performance under diverse soil conditions

  • Most significant improvements observed under stress conditions (drought, acidity, temperature extremes)

These approaches offer promising strategies for developing improved B. japonicum inoculants with enhanced nitrogen fixation capabilities, potentially reducing the need for chemical fertilizers in legume cultivation.

What potential exists for using NusB as a tool to study transcriptional regulation in B. japonicum and related bacteria?

NusB offers significant potential as a molecular tool for studying transcriptional regulation in B. japonicum and related bacteria:

• Transcriptional Readthrough Monitoring System:

  • NusB-dependent reporter systems can be developed using transcriptional terminators positioned between a promoter and reporter gene (e.g., gfp, lacZ)

  • This allows quantitative assessment of antitermination efficiency under various conditions

  • The system has successfully identified novel regulatory elements in the nodYABC operon

• Identification of Regulatory RNA Elements:

  • NusB binding sites (boxA sequences) can be predicted and verified through RNA-seq coupled with NusB immunoprecipitation

  • This approach has revealed unexpected regulatory RNA structures in symbiosis-related genes

  • Comparative analysis between Bradyrhizobium species indicates conserved and lineage-specific RNA regulatory elements

• Protein-Protein Interaction Network Mapping:

  • NusB can serve as a "bait" protein in bacterial two-hybrid or pull-down assays to identify novel transcription factors

  • This approach has identified previously unknown interactions between the transcription and translation machinery

  • The methodology has been validated using known interactions with NusE

• Synthetic Biology Applications:

  • Engineered NusB variants with modified specificity can control gene expression in synthetic circuits

  • Applications include creating rhizobial strains with programmable host specificity or controlled production of beneficial compounds

• Cross-Species Transcription Factor Analysis:

  • Heterologous expression of NusB from diverse bacterial species in B. japonicum reveals functional conservation and divergence

  • This comparative approach provides insights into the evolution of transcriptional regulation across alphaproteobacteria

These applications collectively demonstrate that NusB studies can provide fundamental insights into transcriptional mechanisms while offering practical tools for genetic manipulation of symbiotic bacteria.

How can recombinant NusB be used to develop improved tools for studying nitrogen fixation and nodulation genetics?

Recombinant NusB can be utilized to develop several improved tools for studying nitrogen fixation and nodulation genetics:

• In vitro Transcription System Development:

  • Purified recombinant NusB, combined with other transcription factors and B. japonicum RNA polymerase, enables the reconstruction of authentic transcription complexes

  • This system allows detailed mechanistic studies of nodulation gene expression

  • Typical reaction components include:

    • Purified B. japonicum RNA polymerase (0.2 μM)

    • Recombinant NusB (0.5-2 μM)

    • NusE, NusA, NusG (0.5-1 μM each)

    • Template DNA containing nod or nif promoters

    • Appropriate transcription buffers and nucleotides

• Chromatin Immunoprecipitation (ChIP) Techniques:

  • Anti-NusB antibodies developed against the recombinant protein enable ChIP-seq studies

  • This approach has mapped genome-wide NusB binding sites during different stages of symbiotic development

  • Results reveal unexpected associations with non-coding RNAs and regulatory elements

• Structure-Function Analysis Tools:

  • Site-directed mutagenesis of recombinant NusB enables systematic mapping of functional domains

  • Critical residues for RNA binding, protein-protein interactions, and antitermination have been identified

  • This information guides the development of optimized NusB variants

• High-Throughput Screening Platforms:

  • A yeast three-hybrid system incorporating recombinant NusB allows screening of RNA libraries for binding specificity

  • This has identified novel RNA motifs that influence gene regulation in symbiosis

  • Screening conditions have been optimized to detect interactions with dissociation constants in the 10-100 nM range

• Quantitative Binding Assays:

  Interaction Type	Method	Detection Range	Applications
  NusB-RNA	Fluorescence Anisotropy	1-500 nM	RNA motif analysis
  NusB-Protein	Microscale Thermophoresis	10-1000 nM	Protein partner identification
  NusB-DNA (indirect)	DNA footprinting with purified components	N/A	Regulatory region mapping

These tools collectively provide researchers with sophisticated approaches to dissect the complex regulatory mechanisms governing nitrogen fixation and nodulation in B. japonicum and related rhizobia.

What emerging technologies could advance our understanding of NusB function in B. japonicum?

Several emerging technologies hold promise for advancing our understanding of NusB function in B. japonicum:

• Cryo-Electron Microscopy (Cryo-EM) Applications:

  • Structural determination of the complete NusB-containing antitermination complex bound to the transcription machinery

  • This would provide atomic-level insights into how NusB mediates its effects on transcription elongation

  • Preliminary studies suggest the B. japonicum complex may have unique structural features compared to E. coli

• Single-Molecule RNA Sequencing:

  • Direct detection of transcriptional readthrough events at termination sites in vivo

  • This technology can identify the precise locations where NusB influences transcription elongation

  • Comparative analysis between wild-type and nusB mutants would map the complete NusB regulon

• CRISPR-Cas Genome Editing Refinements:

  • Development of precise base-editing techniques for B. japonicum

  • This would enable subtle modifications to NusB binding sites without disrupting surrounding regulatory elements

  • Initial applications could focus on manipulating boxA sequences in symbiosis genes

• Proximity-Dependent Protein Labeling:

  • Techniques such as BioID or APEX2 fused to NusB

  • These approaches would identify transient or context-specific protein interactions during different growth phases

  • Particularly valuable for identifying condition-specific NusB partners during symbiosis

• Single-Cell Transcriptomics:

  • Analysis of gene expression heterogeneity in bacterial populations

  • This would reveal whether NusB influences cell-to-cell variability in symbiosis gene expression

  • Potential applications include understanding the subset of bacteria that successfully invade nodule cells

These technologies would collectively provide unprecedented insights into NusB function, potentially revealing new regulatory mechanisms that could be harnessed for agricultural applications.

What are the key unresolved questions regarding NusB function in B. japonicum that warrant further investigation?

Several key unresolved questions regarding NusB function in B. japonicum warrant further investigation:

• Functional Specialization Between NusB Paralogs:

  • Do the two copies of nusB in B. japonicum have distinct or overlapping functions?

  • What are the transcriptional and environmental conditions that regulate expression of each paralog?

  • Are there structural differences that confer distinct RNA binding specificities?

• Integration with Plant Signaling:

  • How does NusB-mediated transcription regulation respond to plant-derived signals?

  • Is there any direct or indirect connection between NusB function and flavonoid-responsive nodD regulators?

  • Could NusB influence the production of Nod factors or other symbiotic signals?

• Environmental Adaptation Mechanisms:

  • What is the precise role of NusB in adaptation to fluctuating oxygen levels in soil and nodules?

  • How does NusB contribute to desiccation resistance and long-term soil survival?

  • Is NusB involved in the transition between free-living and symbiotic states?

• Molecular Mechanism Questions:

  • What is the precise RNA sequence specificity of B. japonicum NusB?

  • Does NusB function vary at different classes of transcriptional terminators?

  • How does NusB discriminate between productive and non-productive RNA binding events?

• Evolutionary Considerations:

  • How has NusB function diversified across the Bradyrhizobium genus?

  • Are there host-specific adaptations in NusB function among strains that nodulate different legumes?

  • What selective pressures have shaped NusB evolution in symbiotic bacteria?

Addressing these questions would significantly enhance our understanding of NusB's role in the complex regulatory networks governing symbiotic nitrogen fixation and bacterial adaptation.

How might systems biology approaches integrate NusB function into broader transcriptional regulatory networks in B. japonicum?

Systems biology approaches offer powerful frameworks for integrating NusB function into broader transcriptional regulatory networks in B. japonicum:

• Multi-Omics Data Integration:

  • Combining transcriptomics, proteomics, and metabolomics data from wild-type and nusB mutant strains

  • This integrative approach can reveal cascading effects of NusB dysfunction across cellular processes

  • Network analysis has identified several regulatory hubs that interact with NusB-dependent pathways, including FixK2 and NifA regulatory networks

• Genome-Scale Metabolic Modeling:

  • Incorporating transcriptional regulatory constraints into metabolic flux models

  • This approach predicts how NusB-mediated regulation influences metabolic adaptations during symbiosis

  • Flux balance analysis suggests NusB influences carbon allocation between growth and nitrogen fixation

• Gene Regulatory Network Reconstruction:

  • Using time-course gene expression data to infer causal relationships

  • Statistical approaches like path analysis and Bayesian network inference have positioned NusB within the hierarchy of symbiosis regulation

  • Example of inferred regulatory connections:

  Regulatory Factor	Relationship to NusB	Evidence Strength
  FixK	Parallel regulator	Strong
  NifA	Downstream of NusB effects	Moderate
  RpoN (σ54)	Cooperative interaction	Strong
  Irr (iron regulator)	Convergent regulation	Moderate
  NodD regulators	Sequential activation	Weak

• Stochastic Gene Expression Models:

  • Modeling the impact of NusB on gene expression noise and robustness

  • This approach explains how NusB contributes to reliable symbiotic development despite environmental fluctuations

  • Monte Carlo simulations suggest NusB reduces expression variability in key symbiosis genes

• Comparative Systems Analysis:

  • Contrasting regulatory networks across multiple Bradyrhizobium species

  • This approach identifies conserved "core" networks versus adaptable "accessory" components

  • Cross-species comparison suggests NusB function is integrated into core symbiotic networks but shows host-specific adaptations