The study of bacterial sociality, understanding when and how bacteria interact and the consequences of those interactions, has rapidly grown in popularity over the last 20 years. This has revealed that bacteria engage in a remarkable variety of social interactions, including complex cell-cell communication, the exchange of an arsenal of inhibitory molecules, and the sharing of metabolites. These interactions can have significant consequences for the host or environment they reside in, and numerous mechanisms of interaction have attracted significant interest for their therapeutic potential. The present special issue contains eight review articles addressing three major questions in bacterial sociality (Who is where? How are they interacting? And what are the implications of this?) alongside five methods-focused articles that introduce some particularly valuable approaches for the study of bacterial interactions.

Public microbial genomes encode an immense record of biological diversity, evolution and molecular function, but much of this information remains difficult to reuse because raw sequencing data are not uniformly assembled, quality controlled, annotated or searchable at scale. Here we present AllTheBacteria, an open, community-built resource that transforms public bacterial short-read whole-genome sequencing reads into a uniformly processed discovery platform. The current analysed release contains 2,440,377 high-quality bacterial and archaeal genomes from 11,273 species, together with standardized taxonomic assignments, genome annotations, antimicrobial resistance calls, antiphage-defence annotations, protein structure predictions and AI-ready sequence tables. We show that this infrastructure enables applications that would otherwise be impractical, from global sequence search and outbreak contextualization to pangenome method development, antimicrobial resistance reservoir mapping and antiphage-defence ecology. As a stringent experimental demonstration, we mined 3,919,096 encrypted peptide fragments from AllTheBacteria proteomes using our deep learning model APEX 1.1, identifying 1,867 candidates with predicted antimicrobial activity. We synthesized 24 representative peptides and tested them against 20 clinically relevant bacterial strains, including antibiotic-resistant pathogens. Multiple peptides showed low-micromolar activity, membrane-responsive conformational transitions and selective envelope perturbation. A lead molecule, ATB20, reduced Acinetobacter baumannii burden in a murine skin abscess model with efficacy comparable to polymyxin B and no overt toxicity. Together, these results establish AllTheBacteria as both a foundational community resource for microbiology and a renewable engine for AI-guided antimicrobial discovery.

Antibiotic resistance and the microbiome are two of the most prominent and highly active research areas currently in microbiology. However, these studies are commonly siloed. Research into antibiotic resistance often takes a highly pathogenic-centric view, and microbiome studies typically assess changes in community composition at the genus or species level, rather than at the level of small changes in bacterial genotype that often underpin rapid and significant changes in antibiotic resistance. One of the major mechanisms of antibiotic resistance evolution is via the acquisition of de novo resistance mutations, spontaneous mutations that occur randomly and provide a selective advantage in the presence of antibiotics. In this perspective, we address how interactions within the microbiome can shape the emergence and spread of de novo resistance mutations. We outline existing theoretical and empirical support for how microbial interactions have the potential to influence (i) the probability of de novo resistance mutations emerging, (ii) the fitness costs associated with new resistance mutations and (iii) the long-term selection against resistance and the ability of resistant mutants to transmit to new sites. Existing evolutionary theory may help us predict how microbial interactions will impact the probability of resistance mutations emerging, through understanding how microbial communities will impact pathogen population size, mutation rates and the supply of genetic variation. While a number of these links are simple and intuitive, there is a need for empirical data to understand how the complexity of interactions that exist within a microbial community at any one time come together to shape the evolution of antibiotic resistance. Future research in this field has the potential to inform the development of novel strategies to combat antibiotic resistance based on manipulating microbial interactions.

Plasmids are a ubiquitous feature of bacterial genomes, but the forces driving genes and phenotypes to become associated with plasmids are poorly understood. To address this problem, we compared the fitness effects of chromosomal and plasmid genes in the plant symbiont Rhizobium leguminosarum. The relative abundance of beneficial genes on plasmids was very low compared to the chromosome across niches that reflect key steps in plant colonization. Two lines of evidence support the hypothesis that this pattern emerges because evolutionary processes drive beneficial genes to move from plasmids to the bacterial chromosome. First, weakly beneficial genes that increased fitness in a single niche were evenly distributed between plasmids and the chromosome, whereas the chromosome was enriched for strongly beneficial genes that increased fitness across multiple niches. Second, beneficial genes were more prevalent on recently acquired plasmids compared to ancient plasmids. Our findings support a model in which bacterial lineages initially acquire plasmids due to the beneficial genes that they carry, but the movement of beneficial genes to the chromosome gradually erodes the ecological value of plasmids. These findings reconcile existing models of plasmids and highlight the challenge of understanding how plasmids can persist over the long term.

BACKGROUND:Parasites are foundational to ecosystem health both as indicator species of community productivity and as drivers of diversity. In bacterial communities, bacteriophage viruses can have such roles as they track and modulate the dynamic composition of bacterial hosts within an ecosystem. We aimed to test whether viromes can be used as broad signatures of microbiome health using previously published results across systems. METHODS:In this systematic review and meta-analysis, we searched PubMed, Google Scholar, Scopus, and Web of Science from Jan 22, 2022, to Sept 17, 2024, for peer-reviewed, primary literature published in English, using search terms "phage diversity", "microbiome", "virome", "virus", "phageome", "disease", and "dysbiosis". Inclusion criteria were: a comparison between a dysbiosis state and a healthy state in a human or animal host; a defined host organism and microbiome site; examination of the virome; an obtained measure of virome diversity (α, β, or both); use of statistical analysis to assess whether α or β diversity are changed in dysbiosis; and sufficient methodology description on viral isolation and on virus sequence analysis pipeline. We conducted a qualitative data analysis to assess factors explaining changes to virome diversity in dysbiosis. We then calculated response ratios for each study to test for overall patterns of virome α diversity change under disturbance. Finally, we conducted a quantitative analysis on studies from which we were able to obtain paired virome and bacteriome α diversity data to examine the correlation between these data in defined health compared with defined disturbance conditions. This study was not registered. FINDINGS:We identified a total of 74 studies for inclusion that spanned human (n=61), mouse (n=8), pig (n=3), dog (n=1), and cow (n=1) hosts and a diverse spectrum of infections and diseases. By comparing observed phage and bacterial diversity in microbiomes characterised by dysbiosis with those considered control populations, we were able to identify some key commonalities. Of the 69 studies that investigated changes to α diversity of the virome in dysbiosis, 28 (41%) reported significant changes, but with variable directional change. Of 38 datasets (from 30 studies) for which virome α diversity values were available, 22 (58%) gave a response ratio of less than 1 (α diversity decreases in dysbiosis) and 16 (42%) of more than 1 (α diversity increases in dysbiosis); however, in 27 (71%) datasets, 95% CIs overlapped with 1 (ie, no change in α diversity). We found shifting virome composition to be a more consistent signature of dysbiosis, with 47 (69%) of 68 studies reporting a significant change in viral β diversity with dysbiosis. 62 (89%) of 70 studies reported significant enrichment of system-specific viral taxa under dysbiosis. Our quantitative correlation analysis suggested that bacterial α diversity is a greater predictor of virome α diversity in healthy groups (mean r2=0·380; 95% CI 0·597-0·163) than in dysbiosis (mean r2=0·118, 0·223-0·012; sign test for asymmetric non-parametric data p=4·9 × 10-10). INTERPRETATION:Overall, although specific viral signatures of dysbiosis are likely to be highly disease-specific and condition-specific, we show that existing ecological theory shows promise in predicting the relationship between bacterial and phage diversity and in providing broad signatures of dysbiosis across disease systems. Our observation that the relationship between bacterial and phage diversity breaks down under disturbance suggests that this feature could be a useful signature of dysbiosis and that future studies incorporating the virome could provide opportunity to diagnose, treat, and better understand the causes of microbiome disturbance. FUNDING:There was no funding source for this study.

The emergence and spread of antibiotic resistance in bacterial pathogens is a global health threat. One important unanswered question is how antibiotic resistance influences the ability of a pathogen to invade the host-associated microbiome. Here we investigate how antibiotic resistance impacts the ability of a bacterial pathogen to invade bacteria from the microbiome, using the opportunistic bacterial pathogen Pseudomonas aeruginosa and the respiratory microbiome as our model system. We measure the ability of P. aeruginosa spontaneous antibiotic-resistant mutants to invade pre-established cultures of commensal respiratory microbes in an assay that allows us to link specific resistance mutations with changes in invasion ability. While commensal respiratory microbes tend to provide some degree of resistance to P. aeruginosa invasion, antibiotic resistance is a double-edged sword that can either help or hinder the ability of P. aeruginosa to invade. The directionality of this help or hindrance depends on both P. aeruginosa genotype and respiratory microbe identity. Specific resistance mutations in genes involved in multidrug efflux pump regulation are shown to facilitate the invasion of P. aeruginosa into Staphylococcus lugdunensis, yet impair invasion into Rothia mucilaginosa and Staphylococcus epidermidis. Streptococcus species provide the strongest resistance to P. aeruginosa invasion, and this is maintained regardless of antibiotic resistance genotype. Our study demonstrates how the cost of mutations that provide enhanced antibiotic resistance in P. aeruginosa can crucially depend on community context. We suggest that attempts to manipulate the microbiome should focus on promoting the growth of commensals that can increase the fitness costs associated with antibiotic resistance and provide robust inhibition of both wildtype and antibiotic-resistant pathogen strains.

Parasites are foundational to ecosystem health both as indicator species of community productivity but also as drivers of diversity. In bacterial communities, bacteriophage viruses can play such a role as they track the dynamic composition of bacterial hosts, and in the case of lytic phages, confer a growth advantage to lower abundance bacteria while adapting to more common ones. We set out to test whether viromes can be used as signatures of microbiome health using previously published results across systems. By comparing observed phage and bacterial diversity between microbiomes characterized by disturbance (so-called dysbiosis) and those considered control populations, we were able to identify some key commonalities. While just under half of studies report significant changes in viral species richness in dysbiosis, just under two thirds of studies find the viral composition to shift in dysbiosis, with specific viral taxa enrichment acting as a common signature of dysbiosis. Our analyses also suggest that the positive relationship between bacteriome and virome alpha diversity observed in health breaks down under microbiome disturbance. Overall, while specific viral signatures of dysbiosis are likely to be highly disease- and condition-specific, existing ecological theory shows clear promise in predicting and explaining microbiome health. Future data on bacteria-phage diversity relationships may provide us with much needed opportunity to diagnose, treat, and better understand the causes of dysbiosis. Evidence before this study Being able to identify signatures of microbiome health (or lack thereof) has the potential to improve the way we diagnose and treat disease. To do this, the bacterial microbiome is traditionally characterised at the 16S taxonomic level, and changes in composition are linked to changes in disease status. More recently, the field of viromics has gained attention, and studies have begun to probe the relationship between the virome and health or disturbance (‘dysbiosis’). This work has focused to date on finding single phages that indicate presence of known pathogens, or in a few cases the relationship between viral diversity and disease. To our knowledge, no work has yet sought to identify a common signature of dysbiosis or find commonalities across systems that suggest a role for phages in dysbiosis. Decades of ecological theory has shown how parasites can shape the ecology and evolution of their hosts, and here we argue that bacteriophage viruses have the potential to shape these same processes within microbial communities. The motivation for the current work was thus to ask whether existing ecological theory could help us identify viral signatures of dysbiosis in the microbiome. Added value of this study This study employed a systematic review and meta-analysis to test whether and when phage communities can be used as signatures of microbiome health. To do this, we synthesized previously published results that measure composition of the virome between bacterial microbiomes characterised by health or dysbiosis. We found a total of 39 studies across human, mouse, pig and cow hosts that spanned a diverse spectrum of dysbioses, including bacterial infections, viral infections, and varied diseases such as cancer, cirrhosis, and inflammatory bowel disease, and identified a number of commonalities. Just under half of these studies reported a significant change in viral species richness in dysbiosis, and just under two thirds reported the viral composition to shift in dysbiosis. While the vast majority of studies report an enrichment of specific viral taxa associated with dysbiosis, there is little overlap among studies regarding the identity of these enriched taxa. Finally, our analysis provides evidence that the positive relationship between bacteriome and virome alpha diversity breaks down in dysbiosis. Implications of all available evidence Synthesis of the available evidence suggests that while looking for specific viral taxa as signatures may be limited to associations that are highly disease or condition specific, there is promise for the use of existing ecological theory in predicting and explaining microbiome health when considering compositional changes in the virome. Prospective studies should look to expand the data we have on bacteria-phage relationships at the level of species richness and community compositions, and we argue that more routinely investigating the virome or phageome, in addition to collecting 16S taxonomic descriptions of the microbial community, would help improve our ability to identify signatures of microbiome health. These viral signatures may offer early warning signs of microbiome disturbance and disease. This has clear relevance to our ability to diagnose, treat, and understand the underlying causes of disease. ### Competing Interest Statement The authors have declared no competing interest.

Rhizobial attachment to host legume roots is the first physical interaction of bacteria and plants in symbiotic nitrogen fixation. The pH-dependent primary attachment of Rhizobium leguminosarum biovar viciae 3841 to Pisum sativum (pea) roots was investigated by genome-wide insertion sequencing, luminescence-based attachment assays, and proteomic analysis. Under acid, neutral, or alkaline pH, a total of 115 genes are needed for primary attachment under one or more environmental pH, with 22 genes required for all. These include components of cell surfaces and membranes, together with enzymes that construct and modify them. Mechanisms of dealing with stress also play a part; however, exact requirements vary depending on environmental pH. RNASeq showed that knocking out the two transcriptional regulators required for attachment causes massive changes in the bacterial cell surface. Approximately half of the 54 proteins required for attachment at pH 7.0 have a role in the later stages of nodule formation. We found no evidence for a single rhicadhesin responsible for alkaline attachment, although sonicated cell surface fractions inhibited root attachment at alkaline pH. Our results demonstrate the complexity of primary root attachment and illustrate the diversity of mechanisms involved. IMPORTANCE The first step by which bacteria interact with plant roots is by attachment. In this study, we use a combination of insertion sequencing and biochemical analysis to determine how bacteria attach to pea roots and how this is influenced by pH. We identify several key adhesins, which are molecules that enable bacteria to stick to roots. This includes a novel filamentous hemagglutinin which is needed at all pHs for attachment. Overall, 115 proteins are required for attachment at one or more pHs.

The host-associated microbiome is an important barrier to bacterial pathogen colonization and can mediate protection through a variety of mechanisms. We wanted to investigate the potential consequences of selection imposed by commensal bacterial competitors on an invading bacterial pathogen. To do this, we tested the ability of the opportunistic pathogen Pseudomonas aeruginosa to invade pre-established communities of an abundant commensal bacterium in the human microbiome, Staphylococcus epidermidis . We passaged ten independent lines of P. aeruginosa through daily invasion into a pre-established S. epidermidis population (coculture evolved lines), alongside daily passage through monoculture conditions (monoculture evolved lines). The monoculture evolved lines showed strong parallel evolution in the Wsp (Wrinkly spreader phenotype) signal transducing system involved in biofilm formation, and significantly elevated biofilm formation. On the other hand, adaptation to S. epidermidis occurred via mutations in a diverse set of genes, and the coculture evolved lines showed much weaker evidence for parallel evolution, suggesting that the selective pressure imposed by competition with S. epidermidis is more complex than the pressure imposed by culture conditions. Interestingly, the elevated biofilm formation phenotype seen in the monoculture evolved lines was not observed in the lines evolved in the presence of S. epidermidis , raising the question of whether enhanced biofilm formation did not evolve with S. epidermidis present because it was not beneficial, or because S. epidermidis may be able to restrict this evolutionary path by inhibiting biofilm formation. ### Competing Interest Statement The authors have declared no competing interest.

AbstractThe host-associated microbiome is an important barrier to bacterial pathogen colonization and can mediate protection through a variety of mechanisms. We wanted to investigate the potential consequences of selection imposed by commensal bacterial competitors on an invading bacterial pathogen. To do this, we tested the ability of the opportunistic pathogenPseudomonas aeruginosato invade pre-established communities of an abundant commensal bacterium in the human microbiome,Staphylococcus epidermidis. We passaged ten independent lines ofP. aeruginosathrough daily invasion into a pre-establishedS. epidermidispopulation (coculture evolved lines), alongside daily passage through monoculture conditions (monoculture evolved lines). The monoculture evolved lines showed strong parallel evolution in the Wsp (Wrinkly spreader phenotype) signal transducing system involved in biofilm formation, and significantly elevated biofilm formation. On the other hand, adaptation toS. epidermidisoccurred via mutations in a diverse set of genes, and the coculture evolved lines showed much weaker evidence for parallel evolution, suggesting that the selective pressure imposed by competition withS. epidermidisis more complex than the pressure imposed by culture conditions. Interestingly, the elevated biofilm formation phenotype seen in the monoculture evolved lines was not observed in the lines evolved in the presence ofS. epidermidis, raising the question of whether enhanced biofilm formation did not evolve withS. epidermidispresent because it was not beneficial, or becauseS. epidermidismay be able to restrict this evolutionary path by inhibiting biofilm formation.

Antibiotic resistance poses a global health threat, but the within-host drivers of resistance remain poorly understood. Pathogen populations are often assumed to be clonal within hosts, and resistance is thought to emerge due to selection for de novo variants. Here we show that mixed strain populations are common in the opportunistic pathogen P. aeruginosa. Crucially, resistance evolves rapidly in patients colonized by multiple strains through selection for pre-existing resistant strains. In contrast, resistance evolves sporadically in patients colonized by single strains due to selection for novel resistance mutations. However, strong trade-offs between resistance and growth rate occur in mixed strain populations, suggesting that within-host diversity can also drive the loss of resistance in the absence of antibiotic treatment. In summary, we show that the within-host diversity of pathogen populations plays a key role in shaping the emergence of resistance in response to treatment.

AbstractThe emergence and spread of antibiotic resistance in bacterial pathogens is a global health threat. One important unanswered question is how antibiotic resistance influences the ability of a pathogen to invade the host-associated microbiome. Here we investigate how antibiotic resistance impacts the ability of the opportunistic bacterial pathogenPseudomonas aeruginosato invade the respiratory microbiome, by measuring the ability ofP. aeruginosaspontaneous antibiotic resistant mutants to invade pre-established cultures of commensal respiratory microbes. We find that commensal respiratory microbes tend to inhibit the growth ofP. aeruginosa, and antibiotic resistance is a double-edged sword that can either help or hinder the ability ofP. aeruginosato overcome this inhibition. The directionality of this help or hinderance depends on bothP. aeruginosagenotype and respiratory microbe identity. Antibiotic resistance facilitates the invasion ofP. aeruginosaintoStaphylococcus lugdunensis,yet impairs invasion intoRothia mucilaginosaandStaphylococcus epidermidis.Streptococcusspecies provide the strongest inhibition toP. aeruginosainvasion, and this is maintained regardless of antibiotic resistance genotype. Our study demonstrates how antibiotic resistance can alter the ability of a bacterial pathogen to invade the respiratory microbiome and suggests that attempts to manipulate the microbiome should focus on promoting the growth of commensals that can provide robust inhibition of both wildtype and antibiotic resistant pathogen strains.

Infections that involve interkingdom microbial communities, such as those between bacteria and yeast pathogens, are difficult to treat, associated with worse patient outcomes, and may be a source of antimicrobial resistance. In this review, we address co-occurrence and co-infections of Candida albicans and Pseudomonas aeruginosa, two pathogens that occupy multiple infection niches in the human body, especially in immunocompromised patients. The interaction between the pathogen species influences microbe-host interactions, the effectiveness of antimicrobials and even infection outcomes, and may thus require adapted treatment strategies. However, the molecular details of bacteria-fungal interactions both inside and outside the infection sites, are insufficiently understanding the P. aeruginosa-C. albicans interaction network through integrated systems biology approaches will capture the highly dynamic and complex nature of these polymicrobial infections and lead to a more comprehensive understanding of clinical observations such as reshaped immune defences and low antimicrobial treatment efficacy.

Women are underrepresented in senior academic positions within microbiology globally. Studies show that gender bias affects the progression of women in academia, but there is evidence that improving conscious awareness of bias can improve equity in this regard. Here we analyse the publication data associated with review articles within the microbiology field to investigate the statistical associations with author gender. We analyse the data from review articles published between 2010 and 2022 in three leading microbiology review journals: Nature Reviews Microbiology, Trends in Microbiology and Annual Review of Microbiology. We find a significant association between the gender of the lead author and the gender of co-authors in multi-author publications. Review articles with men lead authors have a significantly reduced proportion of women co-authors compared to reviews with women lead authors. Given the existing differences in the proportions of men and women in lead author positions, this association may have important consequences for the relative visibility of women in microbiology, along with negative impacts on scientific output relating to reduced collaboration diversity.

Women are underrepresented in senior academic positions within microbiology globally. Studies show that gender bias affects the progression of women in academia, but there is evidence that improving conscious awareness of bias can improve equity in this regard. Here we carry out a bibliometric analysis of review articles within the microbiology field to investigate the statistical associations with author gender. We analyse the publication data from 1857 review articles published between 2010 and 2022 in three leading microbiology review journals: Nature Reviews Microbiology, Trends in Microbiology, and Annual Review of Microbiology. We find a significant association between the gender of the lead author and the gender of co-authors in multi-author publications. Review articles with men lead authors have a significantly reduced proportion of women co-authors compared to reviews with women lead authors. Given the existing differences in the proportions of men and women in lead author positions, this association may have important consequences for the relative visibility of women in microbiology, along with potential negative impacts on scientific output relating to reduced collaboration diversity. We further probe associations between gender and citation metrics, acknowledgement of contributions, and publishing during the Covid-19 pandemic within microbiology reviews.

Bacteria have the potential to migrate between sites in the human body, but the dynamics and consequences of within-host translocation remain poorly understood. Here we investigate the link between gut and lung Pseudomonas aeruginosa populations in an intensively sampled ICU patient using a combination of genomics, isolate phenotyping, host immunity profiling, and clinical data. Crucially, we show that lung colonization was driven by the repeated translocation of bacterial clones from the gut. Meropenem treatment for a suspected urinary tract infection selected for elevated resistance in both the gut and lung. However, resistance was driven by parallel evolution and organ-specific selective pressures, and within-host transmission had only a minor impact on AMR. These findings suggest that reducing intestinal colonization of Pseudomonas may be an effective way to prevent lung infections in critically ill patients.

Bacterial pathogens show high levels of chromosomal genetic diversity, but the influence of this diversity on the evolution of antibiotic resistance by plasmid acquisition remains unclear. Here, we address this problem in the context of colistin, a ‘last line of defence’ antibiotic. Using experimental evolution, we show that a plasmid carrying the MCR-1 colistin resistance gene dramatically increases the ability of Escherichia coli to evolve high-level colistin resistance by acquiring mutations in lpxC, an essential chromosomal gene involved in lipopolysaccharide biosynthesis. Crucially, lpxC mutations increase colistin resistance in the presence of the MCR-1 gene, but decrease the resistance of wild-type cells, revealing positive sign epistasis for antibiotic resistance between the chromosomal mutations and a mobile resistance gene. Analysis of public genomic datasets shows that lpxC polymorphisms are common in pathogenic E. coli, including those carrying MCR-1, highlighting the clinical relevance of this interaction. Importantly, lpxC diversity is high in pathogenic E. coli from regions with no history of MCR-1 acquisition, suggesting that pre-existing lpxC polymorphisms potentiated the evolution of high-level colistin resistance by MCR-1 acquisition. More broadly, these findings highlight the importance of standing genetic variation and plasmid/chromosomal interactions in the evolutionary dynamics of antibiotic resistance.

Viruses of bacteria (bacteriophages or phage) have broad effects on bacterial ecology and evolution in nature that mediate microbial interactions, shape bacterial diversity, and influence nutrient cycling and ecosystem function. The unrelenting impact of phages within the microbial realm is the result, in large part, of their ability to rapidly evolve in response to bacterial host dynamics. The knowledge gained from laboratory systems, typically using pairwise interactions between single-host and single-phage systems, has made clear that phages coevolve with their bacterial hosts rapidly, somewhat predictably, and primarily by counteradapting to host resistance. Recent advancement in metagenomics approaches, as well as a shifting focus toward natural microbial communities and host-associated microbiomes, is beginning to uncover the full picture of phage evolution and ecology within more complex settings. As these data reach their full potential, it will be critical to ask when and how insights gained from studies of phage evolution in vitro can be meaningfully applied to understanding bacteria-phage interactions in nature. In this review, we explore the myriad ways that phagesshape and are themselves shaped by bacterial host populations and communities, with a particular focus on observed and predicted differences between the laboratory and complex microbial communities.

AbstractPrimary attachment of rhizobia to host legume roots depends on pH and is the first physical interaction during nodulation. Genome-wide insertion sequencing, luminescence-based attachment assays and proteomic analysis demonstrate primary attachment ofRhizobium leguminosarumbiovarviciae3841 toPisum sativum(pea) roots is more complex than previously thought. In total, 115 proteins are needed for initial attachment under one or more test conditions (acid, neutral or alkaline pH), with 22 required under all conditions. These include cell-surface filamentous hemagglutinin adhesin (RL4382) and its transporter (RL4381), transmembrane protein RL2400, RL3752 (PssA, glycosyl transferase) affecting capsular polysaccharide and transcriptional regulator RL4145 (PckR). RNASeq was used to determine targets of RL4145 (PckR) and regulator RL3453. The 54 proteins required for attachment at pH 7.0 were investigated for nodulation phenotypes. Glucomannan biosynthesis protein A (GmsA) is needed at pH 6.5 and pH 7.0. Membrane proteins DgkA and ImpA are required specifically at pH 6.5, and RpoZ at pH 7.5. Sonicated cell surface fractions inhibited root attachment at alkaline pH but no overlap between proteins identified by proteomic and INseq analysis, suggests there is no single rhicadhesin needed for alkaline attachment. Our results demonstrate the complexity of primary root attachment and diversity of mechanisms involved.