Light cues induce protective anticipation of environmental water loss in terrestrial bacteria.

The ecological significance of light perception in nonphotosynthetic bacteria remains largely elusive. In terrestrial environments, diurnal oscillations in light are often temporally coupled to other environmental changes, including increased temperature and evaporation. Here, we report that light functions as an anticipatory cue that triggers protective adaptations to tolerate a future rapid loss of environmental water. We demonstrate this photo-anticipatory stress tolerance in leaf-associated Pseudomonas syringae pv. syringae (Pss) and other plant- and soil-associated pseudomonads. We found that light influences the expression of 30% of the Pss genome, indicating that light is a global regulatory signal, and this signaling occurs almost entirely via a bacteriophytochrome photoreceptor that senses red, far-red, and blue wavelengths. Bacteriophytochrome-mediated light control disproportionally up-regulates water-stress adaptation functions and confers enhanced fitness when cells encounter light prior to water limitation. Given the rapid speed at which water can evaporate from leaf surfaces, such anticipatory activation of a protective response enhances fitness beyond that of a reactive stress response alone, with recurring diurnal wet-dry cycles likely further amplifying the fitness advantage over time. These findings demonstrate that nonphotosynthetic bacteria can use light as a cue to mount an adaptive anticipatory response against a physiologically unrelated but ecologically coupled stress.

An atypical 3-ketoacyl ACP synthase III required for acyl homoserine lactone synthesis in Pseudomonas syringae pv. syringae B728a.

The last step of the initiation phase of fatty acid biosynthesis in most bacteria is catalyzed by the 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). Pseudomonas syringae pv. syringae strain B728a encodes two FabH homologs, Psyr_3467 and Psyr_3830, which we designated PssFabH1 and PssFabH2, respectively. Here, we explored the roles of these two 3-ketoacyl-ACP synthase (KAS) III proteins. We found that PssFabH1 is similar to the Escherichia coli FabH in using acetyl-acetyl-coenzyme A (CoA ) as a substrate in vitro, whereas PssFabH2 uses acyl-CoAs (C4-C10) or acyl-ACPs (C6-C10). Mutant analysis showed that neither KAS III protein is essential for the de novo fatty acid synthesis and cell growth. Loss of PssFabH1 reduced the production of an acyl homoserine lactone (AHL) quorum-sensing signal, and this production was partially restored by overexpressing FabH homologs from other bacteria. AHL production was also restored by inhibiting fatty acid elongation and providing exogenous butyric acid. Deletion of PssFabH1 supports the redirection of acyl-ACP toward biosurfactant synthesis, which in turn enhances swarming motility. Our study revealed that PssFabH1 is an atypical KAS III protein that represents a new KAS III clade that functions in providing a critical fatty acid precursor, butyryl-ACP, for AHL synthesis.IMPORTANCEAcyl homoserine lactones (AHLs) are important quorum-sensing compounds in Gram-negative bacteria. Although their formation requires acylated acyl carrier proteins (ACPs), how the acylated intermediate is shunted from cellular fatty acid synthesis to AHL synthesis is not known. Here, we provide in vivo evidence that Pseudomonas syringae strain B728a uses the enzyme PssFabH1 to provide the critical fatty acid precursor butyryl-ACP for AHL synthesis. Loss of PssFabH1 reduces the diversion of butyryl-ACP to AHL, enabling the accumulation of acyl-ACP for synthesis of biosurfactants that contribute to bacterial swarming motility. We report that PssFabH1 and PssFabH2 each encode a 3-ketoacyl-acyl carrier protein synthase (KAS) III in P. syringae B728a. Whereas PssFabH2 is able to function in redirecting intermediates from ß-oxidation to fatty acid synthesis, PssFabH1 is an atypical KAS III protein that represents a new KAS III clade based on its sequence, non-involvement in cell growth, and novel role in AHL synthesis.

From microbes to microbiomes: Applications for plant health and sustainable agriculture.

Plant-microbe interaction research has had a transformative trajectory, from individual microbial isolate studies to comprehensive analyses of plant microbiomes within the broader phytobiome framework. Acknowledging the indispensable role of plant microbiomes in shaping plant health, agriculture, and ecosystem resilience, we underscore the urgent need for sustainable crop production strategies in the face of contemporary challenges. We discuss how the synergies between advancements in 'omics technologies and artificial intelligence can help advance the profound potential of plant microbiomes. Furthermore, we propose a multifaceted approach encompassing translational considerations, transdisciplinary research initiatives, public-private partnerships, regulatory policy development, and pragmatic expectations for the practical application of plant microbiome knowledge across diverse agricultural landscapes. We advocate for strategic collaboration and intentional transdisciplinary efforts to unlock the benefits offered by plant microbiomes and address pressing global issues in food security. By emphasizing a nuanced understanding of plant microbiome complexities and fostering realistic expectations, we encourage the scientific community to navigate the transformative journey from discoveries in the laboratory to field applications. As companies specializing in agricultural microbes and microbiomes undergo shifts, we highlight the necessity of understanding how to approach sustainable agriculture with site-specific management solutions. While cautioning against over-promising, we underscore the excitement of exploring the many impacts of microbiome-plant interactions. We emphasize the importance of collaborative endeavors with societal partners to accelerate our collective capacity to harness the diverse and yet-to-be-discovered beneficial activities of plant microbiomes.

First report of cucurbit yellow vine disease caused by Serratia marcescens on cucurbit crops in Iowa.

Cucurbit yellow vine disease (CYVD) is caused by Serratia marcescens, vectored by squash bugs (Anasa tristis), and is an emerging disease in many parts of the U.S. CYVD can cause 100% yield losses in cucurbits (Bruton et al., 2003). In the summer of 2021, at the Iowa State University Horticultural Research Station (HRS) in Gilbert, Iowa, we observed leaf yellowing, vine decline, and honey-brown discoloration of the phloem of acorn squash (Cucurbita pepo cv. Table Ace) plants in research fields that were infested with squash bugs. In 2022, we observed similar symptoms on pumpkin (Cucurbita maxima cv. Howden) and muskmelon (Cucumis melo cv. Athena) in different fields at the HRS and on giant pumpkins (Cucurbita maxima cv. Prizewinner) in Jones and Ringgold counties. For up to 3 symptomatic plants of each cucurbit species per location, a 20-cm-long stem section immediately above the soil line was excised, surface sterilized by immersion in 10% sodium hypochlorite and 70% ethanol for 2 min each, then triple rinsed in sterile water. The interior of the cross-section tissue was blotted on Luria agar amended with cycloheximide (100 µg/ml) and tetracycline (20 µg/ml) (Stock et al. 2003). Whitish translucent colonies developed after incubation at 28°C for 48 h. The genomic DNA of three isolates from symptomatic plants of muskmelon (MK01), pumpkin (HFP01), and giant pumpkin (AP01), was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD). S. marcescens species-specific primers YV1 (5'-GGGACTTGCTCCCCGG-3') and YV4 (5'-AACGTCAATTGATGAACGTATTAAGT-3') (Bruton et al. 2003) were used to amplify part of the 16S rDNA gene, and the primers specific to S. marcescens CYVD strains A79F/A79R (Zhang et al., 2005) were used to amplify part of a major facilitatory superfamily (MFS) transporter gene strain. The sequences of the 16S rRNA PCR product for the three isolates were identical and were deposited in NCBI under Accession OR963533. They shared 100% (395/395 nt) identity with other CYVD strains (Rascoe et al. 2003) and those of other S. marcescens strains in NCBI. The sequences of the amplified region of the MFS transporter gene of the three isolates (NCBI Accession OR962261) were identical and showed a 98.8% (319/323 nt) identity to that of non-CYVD-causing S. marcescens strains, such as N10A28 (Accession CP033623.1). Koch's postulates were fulfilled by inoculating C. pepo cv. Zephyr plants with either strain HFP01 or phosphate buffer saline (PBS) (10 plants per treatment) 1 wk after seeding by injecting 300 µl of bacteria (~108 CFU/ml) or PBS using a syringe needle. Plants were incubated at 28°C in a growth room for 4 wks. CYVD symptoms similar to those observed in the field developed on 7 out of 10 plants inoculated with strain HFP01 in one study, and 9 out 10 plants in a replicate study, with none of the PBS-inoculated plants showing CYVD symptoms. Bacteria were isolated from the symptomatic plants with selection on tetracycline. The PCR fragments amplified with YV1/YV4 and A79F/A79R were the same size as those of the pre-inoculation strain HFP01. To our knowledge this is the first report of CYVD in Iowa and in the Upper Midwest of the U.S. CYVD is a devastating disease that poses a significant threat to cucurbit production. This report can serve as an alert for the region's growers and for the development of effective management practices.

Expression and Functional Analysis of the Type III Secretion System Effector Repertoire of the Xylem Pathogen Erwinia tracheiphila on Cucurbits.

The predicted repertoire of type III secretion system effectors (T3SEs) in Erwinia tracheiphila, causal agent of cucurbit bacterial wilt, is much larger than in xylem pathogens in the closely related genera Erwinia and Pantoea. The genomes of strains BHKY and SCR3, which represent distinct E. tracheiphila clades, encode at least 6 clade-specific and 12 shared T3SEs. The strains expressed the majority of the T3SE genes examined in planta. Among the shared T3SE genes, eop1 was expressed most highly in both strains in squash (Cucurbita pepo) and muskmelon (Cucumis melo) but the clade-specific gene avrRpm2 was expressed 40- to 900-fold more than eop1 in BHKY. The T3SEs AvrRpm2, Eop1, SrfC, and DspE contributed to BHKY virulence on squash and muskmelon, as shown using combinatorial mutants involving six T3SEs, whereas OspG and AvrB4 contributed to BHKY virulence only on muskmelon, demonstrating host-specific virulence functions. Moreover, Eop1 was functionally redundant with AvrRpm2, SrfC, OspG, and AvrB4 in BHKY, and BHKY mutants lacking up to five effector genes showed similar virulence to mutants lacking only two genes. The T3SEs OspG, AvrB4, and DspE contributed additively to SCR3 virulence on muskmelon and were not functionally redundant with Eop1. Rather, loss of eop1 and avrB4 restored wild-type virulence to the avrB4 mutant, suggesting that Eop1 suppresses a functionally redundant effector in SCR3. These results highlight functional differences in effector inventories between two E. tracheiphila clades, provide the first evidence of OspG as a phytopathogen effector, and suggest that Eop1 may be a metaeffector influencing virulence. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The Contributions to Virulence of the Effectors Eop1 and DspE Differ Between Two Clades of Erwinia tracheiphila Strains.

Strains of Erwinia tracheiphila, causal agent of bacterial wilt of cucurbits, are divided into distinct clades. Et-melo clade strains wilt Cucumis spp. but not Cucurbita spp., thus exhibiting host specificity, whereas Et-C1 clade strains wilt Cucurbita spp. more rapidly than Cucumis melo, thus exhibiting a host preference. This study investigated the contribution of the effector proteins Eop1 and DspE to E. tracheiphila pathogenicity and host adaptation. Loss of eop1 did not enable Et-melo strains to infect squash (Cucurbita pepo) or an Et-C1 strain to induce a more rapid wilt of muskmelon (Cucumis melo), indicating that Eop1 did not function in host specificity or preference as in the related pathogen E. amylovora. However, overexpression of eop1 from Et-melo strain MDCuke but not from Et-C1 strain BHKY increased the virulence of a BHKY eop1 deletion mutant on muskmelon, demonstrating that the Eop1 variants in the two clades are distinct in their virulence functions. Loss of dspE from Et-melo strains reduced but did not eliminate virulence on hosts muskmelon and cucumber, whereas loss of dspE from an Et-C1 strain eliminated pathogenicity on hosts squash, muskmelon, and cucumber. Thus, the centrality of DspE to virulence differs in the two clades. Et-melo mutants lacking the chaperone DspF exhibited similar virulence to mutants lacking DspE, indicating that DspF is the sole chaperone for DspE in E. tracheiphila, unlike in E. amylovora. Collectively, these results provide the first functional evaluation of effectors in E. tracheiphila and demonstrate clade-specific differences in the roles of Eop1 and DspE.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Biological Control and Microbial Ecology Draft Genome Sequence Data of Glutamicibacter sp. FBE-19, a Bacterium Antagonistic to the Plant Pathogen Erwinia tracheiphila.

Glutamicibacter sp. FBE-19 was isolated based on its strong antagonism to the cucurbit bacterial blight pathogen Erwinia tracheiphila on plates. Members of the Glutamicibacter genus can promote plant growth under saline conditions and antagonize fungi on plates via chitinolytic activity; however, their production of antibacterial compounds has not been examined. Here, we report the genome sequence of strain FBE-19. The genome is 3.85 Mbp with a G+C content of 60.1% and comprises 3,791 genes. Genes that may contribute to its antagonistic activity include genes for the secondary metabolites stenothricin, salinosporamide A, a second ß-lactone compound, and a carotenoid. The Glutamicibacter sp. FBE-19 genome data may be a useful resource if this strain proves to be an effective biocontrol agent against E. tracheiphila.

Characterization of Erwinia tracheiphila Bacteriophage FBB1 Isolated from Spotted Cucumber Beetles that Vector E. tracheiphila.

Erwinia tracheiphila, the causal pathogen of bacterial wilt of cucurbit crops, is disseminated by cucumber beetles. A bacteriophage, designated FBB1 (Fu-Beattie-Beetle-1), was isolated from spotted cucumber beetles (Diabrotica undecimpunctata) that were collected from a field in which E. tracheiphila is endemic. FBB1 was classified into the Myoviridae family based on its morphology, which includes an elongated icosahedral head (106 × 82 nm) and a putatively contractile tail (120 nm). FBB1 infected all 62 E. tracheiphila strains examined and three Pantoea spp. strains. FBB1 virions were stable at 55°C for 1 h and tolerated a pH range from 3 to 12. FBB1 has a genome of 175,994 bp with 316 predicted coding sequences and a GC content of 36.5%. The genome contains genes for a major bacterial outer-membrane protein, a putative exopolysaccharide depolymerase, and 22 predicted transfer RNAs. The morphology and genome indicate that FBB1 is a T4-like virus and thus in the Tevenvirinae subfamily. FBB1 is the first virulent phage of E. tracheiphila to be reported and, to date, is one of only two bacteriophages to be isolated from insect vectors of phytopathogens. Collectively, the results support FBB1 as a promising candidate for biocontrol of E. tracheiphila based on its virulent (lytic) rather than lysogenic lifestyle, its infection of all E. tracheiphila strains examined to date, and its infection of a few nonpathogenic bacteria that could be used to support phage populations when pathogen numbers are low.

Transcriptional responses of Pseudomonas syringae to growth in epiphytic versus apoplastic leaf sites.

Some strains of the foliar pathogen Pseudomonas syringae are adapted for growth and survival on leaf surfaces and in the leaf interior. Global transcriptome profiling was used to evaluate if these two habitats offer distinct environments for bacteria and thus present distinct driving forces for adaptation. The transcript profiles of Pseudomonas syringae pv. syringae B728a support a model in which leaf surface, or epiphytic, sites specifically favor flagellar motility, swarming motility based on 3-(3-hydroxyalkanoyloxy) alkanoic acid surfactant production, chemosensing, and chemotaxis,indicating active relocation primarily on the leaf surface. Epiphytic sites also promote high transcript levels for phenylalanine degradation, which may help counteract phenylpropanoid-based defenses before leaf entry. In contrast, intercellular, or apoplastic,sites favor the high-level expression of genes for GABA metabolism (degradation of these genes would attenuate GABA repression of virulence) and the synthesis of phytotoxins, two additional secondary metabolites, and syringolin A. These findings support roles for these compounds in virulence, including a role for syringolin A in suppressing defense responses beyond stomatal closure. A comparison of the transcriptomes from in planta cells and from cells exposed to osmotic stress, oxidative stress, and iron and nitrogen limitation indicated that water availability, in particular,was limited in both leaf habitats but was more severely limited in the apoplast than on the leaf surface under the conditions tested. These findings contribute to a coherent model of the adaptations of this widespread bacterial phytopathogen to distinct habitats within its host.

Bacterial Wilt of Cucurbits: Resurrecting a Classic Pathosystem.

Bacterial wilt threatens cucurbit crop production in the Midwestern and Northeastern United States. The pathogen, Erwinia tracheiphila, is a xylem-limited bacterium that affects most commercially important cucurbit species, including muskmelon, cucumber, and squash. Bacterial wilt is transmitted and overwintered by striped and spotted cucumber beetles. Since there are few commercially available resistant cultivars, disease management usually relies on use of insecticides to suppress vector populations. Although bacterial wilt was initially described more than 100 years ago, our knowledge of disease ecology and epidemiology advanced slowly for most of the 20th century. However, a recent wave of research has begun to fill in missing pieces of the bacterial wilt puzzle. This article-the first review of research toward understanding the cucurbit bacterial wilt pathosystem-recounts early findings and updates our understanding of the disease cycle, including pathogen and vector biology. We also highlight research areas that could lead to more efficient and ecologically based management of bacterial wilt.

The widespread plant-colonizing bacterial species Pseudomonas syringae detects and exploits an extracellular pool of choline in hosts.

The quaternary ammonium compound (QAC) choline is a major component of membrane lipids in eukaryotes and, if available to microbial colonists of plants, could provide benefits for growth and protection from stress. Free choline is found in homogenized plant tissues, but its subcellular location and availability to plant microbes are not known. Whole-cell bacterial bioreporters of the phytopathogen Pseudomonas syringae were constructed that couple a QAC-responsive transcriptional fusion with well-characterized bacterial QAC transporters. These bioreporters demonstrated the presence of abundant free choline compounds released from germinating seeds and seedlings of the bean Phaseolus vulgaris, and a smaller but consistently detectable amount of QACs, probably choline, from leaves. The localization of P. syringae bioreporter cells to the surface and intercellular sites of plant tissues demonstrated the extracellular location of these QAC pools. Moreover, P. syringae mutants that were deficient in the uptake of choline compounds exhibited reduced fitness on leaves, highlighting the importance of extracellular choline to P. syringae on leaves. Our data support a model in which this choline pool is derived from the phospholipid phosphatidylcholine through plant-encoded phospholipases that release choline into the intercellular spaces of plant tissues, such as for membrane lipid recycling. The consequent extracellular release of choline compounds enables their interception and exploitation by plant-associated microbes, and thus provides a selective advantage for microbes such as P. syringae that are adapted to maximally exploit choline.

Glycine betaine catabolism contributes to Pseudomonas syringae tolerance to hyperosmotic stress by relieving betaine-mediated suppression of compatible solute synthesis.

Many bacteria can accumulate glycine betaine for osmoprotection and catabolize it as a growth substrate, but how they regulate these opposing roles is poorly understood. In Pseudomonas syringae B728a, expression of the betaine catabolism genes was reduced by an osmotic upshift to an intermediate stress level, consistent with betaine accumulation, but was increased by an upshift to a high stress level, as confirmed by an accompanying increase in degradation of radiolabeled betaine. Deletion of the gbcAB betaine catabolism genes reduced osmotolerance at a high osmolarity, and this reduction was due to the relief of betaine-mediated suppression of compatible solute synthesis. This conclusion was supported by the findings that, at high osmolarity, the ΔgbcAB mutant accumulated high betaine levels and low endogenous solutes and exhibited reduced expression of the solute synthesis genes. Moreover, the ΔgbcAB mutant and a mutant deficient in the synthesis of the compatible solutes NAGGN and trehalose exhibited similar reductions in osmotolerance and also in fitness on bean leaves. Activation of betaine catabolism at high osmotic stress resulted, in part, from induction of gbdR, which encodes the transcriptional activator GbdR. Betaine catabolism was subject to partial repression by succinate under hyperosmotic stress conditions, in contrast to strong repression in the absence of stress, suggesting that betaine functions both in nutrition and as an intracellular signal modulating solute synthesis under hyperosmotic stress conditions. Collectively, these results begin to provide a detailed mechanistic understanding of how P. syringae transitions from reliance on exogenously derived betaine to the use of endogenous solutes during adaptation to hyperosmotic conditions.

Physiological and transcriptional responses to osmotic stress of two Pseudomonas syringae strains that differ in epiphytic fitness and osmotolerance.

The foliar pathogen Pseudomonas syringae is a useful model for understanding the role of stress adaptation in leaf colonization. We investigated the mechanistic basis of differences in the osmotolerance of two P. syringae strains, B728a and DC3000. Consistent with its higher survival rates following inoculation onto leaves, B728a exhibited superior osmotolerance over DC3000 and higher rates of uptake of plant-derived osmoprotective compounds. A global transcriptome analysis of B728a and DC3000 following an osmotic upshift demonstrated markedly distinct responses between the strains; B728a showed primarily upregulation of genes, including components of the type VI secretion system (T6SS) and alginate biosynthetic pathways, whereas DC3000 showed no change or repression of orthologous genes, including downregulation of the T3SS. DC3000 uniquely exhibited improved growth upon deletion of the biosynthetic genes for the compatible solute N-acetylglutaminylglutamine amide (NAGGN) in a minimal medium, due possibly to NAGGN synthesis depleting the cellular glutamine pool. Both strains showed osmoreduction of glnA1 expression, suggesting that decreased glutamine synthetase activity contributes to glutamate accumulation as a compatible solute, and both strains showed osmoinduction of 5 of 12 predicted hydrophilins. Collectively, our results demonstrate that the superior epiphytic competence of B728a is consistent with its strong osmotolerance, a proactive response to an osmotic upshift, osmoinduction of alginate synthesis and the T6SS, and resiliency of the T3SS to water limitation, suggesting sustained T3SS expression under the water-limited conditions encountered during leaf colonization.

Ornamental tobacco floral nectar is a rich source of antimicrobial peptides.

Although floral nectar is a rich source of nutrients, it is rarely infected by microorganisms. Defense molecules such as proteins have been identified in this fluid, but defense peptides have been largely overlooked. Thus, the aim of this study was to perform an extensive peptidomic analysis of the ornamental tobacco floral nectar to seek peptides involved in nectar defense. Using LC-MS/MS, 793 peptides were sequenced and characterized. After extensive bioinformatics analysis, six peptides were selected for further characterization, synthesis, and evaluation of their antimicrobial properties against phytopathogenic fungi and bacteria. All six peptides had antimicrobial activity to some extent. However, the activity varied by peptide concentration and microorganism tested. An analysis of the action mechanism revealed damage in the cell membrane induced by peptides. The results show that floral nectar is rich in peptides and that, together with proteins and hydrogen peroxide, they contribute to plant defense against microorganisms during pollination.

Roles of three transporters, CbcXWV, BetT1, and BetT3, in Pseudomonas aeruginosa choline uptake for catabolism.

Pseudomonas aeruginosa uses the quaternary amine choline as a carbon source, osmoprotectant, and macromolecular precursor. The importance of choline in P. aeruginosa physiology is highlighted by the presence of multiple known and putative choline transporters encoded within its genome. This report describes the relative roles of three choline transporters, the ABC transporter CbcXWV and two symporters, BetT1 and BetT3, in P. aeruginosa growth on choline under osmotic conditions that are physiologically relevant to eukaryotic hosts. The increased lag phases exhibited by the ΔbetT1 and ΔbetT1 ΔbetT3 mutants relative to the wild type upon transfer to medium with choline as a sole carbon source suggested roles for BetT1 and BetT3 in cells newly exposed to choline. BetT3 and CbcXWV, but not BetT1, were sufficient to support growth on choline. betT1 and betT3 expression was regulated by the repressor BetI and choline, whereas cbcXWV expression was induced by the activator GbdR and glycine betaine. The data support a model in which, upon transfer to a choline-based medium, the glycine betaine derived from choline taken up by BetT1 and BetT3 promotes subsequent GbdR-mediated cbcXWV induction. Furthermore, growth data indicated that the relative contributions of each transporter varied under different conditions, as BetT1 and CbcXWV were the primary choline transporters under hypo-osmolar conditions whereas BetT3 was the major choline transporter under hyperosmolar conditions. This work represents the first systematic approach to unravel the mechanisms of choline uptake in P. aeruginosa, which has the most complex bacterial choline uptake systems characterized to date.

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.