Elongation of many rod-shaped bacteria occurs by peptidoglycan synthesis at discrete foci along the sidewall of the cells. However, within the Rhizobiales, there are many budding bacteria, in which new cell growth is constrained to a specific region. The phylogeny of the Rhizobiales indicates that this mode of zonal growth may be ancestral. We demonstrate that the rod-shaped bacterium Agrobacterium tumefaciens grows unidirectionally from the new pole generated after cell division and has an atypical peptidoglycan composition. Polar growth occurs under all conditions tested, including when cells are attached to a plant root and under conditions that induce virulence. Finally, we show that polar growth also occurs in the closely related bacteria Sinorhizobium meliloti , Brucella abortus , and Ochrobactrum anthropi . We find that unipolar growth is an ancestral and conserved trait among the Rhizobiales, which includes important mutualists and pathogens of plants and animals.

Summary: Burkholderia glumae BGR1 produces a broad‐host range phytotoxin, called toxoflavin, which is a key pathogenicity factor in rice grain rot and wilt in many field crops. Our molecular and genetic analyses of toxoflavin‐deficient mutants demonstrated that gene clusters for toxoflavin production consist of four transcriptional units. The toxoflavin biosynthesis genes were composed of five genes, toxA to toxE, as Suzuki et al. (2004) reported previously. Genes toxF to toxI, which are responsible for toxoflavin transport, were polycistronic and similar to the genes for resistance‐nodulation‐division (RND) efflux systems. Using Tn3‐gusA reporter fusions, we found that ToxR, a LysR‐type regulator, regulates both the toxABCDE and toxFGHI operons in the presence of toxoflavin as a coinducer. In addition, the expression of both operons required a transcriptional activator, ToxJ, whose expression is regulated by quorum sensing. TofI, a LuxI homologue, was responsible for the biosynthesis of both N‐hexanoyl homoserine lactone and N‐octanoyl homoserine lactone (C8‐HSL). C8‐HSL and its cognate receptor TofR, a LuxR homologue, activated toxJ expression. This is the first report that quorum sensing is involved in pathogenicity by the regulation of phytotoxin biosynthesis and its transport in plant pathogenic bacteria.

Pseudomonas fluorescens B16 is a plant growth-promoting rhizobacterium. To determine the factors involved in plant growth promotion by this organism, we mutagenized wild-type strain B16 using ΩKm elements and isolated one mutant, K818, which is defective in plant growth promotion, in a rockwool culture system. A cosmid clone, pOK40, which complements the mutant K818, was isolated from a genomic library of the parent strain. Tn3-gusA mutagenesis of pOK40 revealed that the genes responsible for plant growth promotion reside in a 13.3-kb BamHI fragment. Analysis of the DNA sequence of the fragment identified 11 putative open reading frames, consisting of seven known and four previously unidentified pyrroloquinoline quinone (PQQ) biosynthetic genes. All of the pqq genes showed expression only in nutrient-limiting conditions in a PqqH-dependent manner. Electrospray ionization-mass spectrometry analysis of culture filtrates confirmed that wild-type B16 produces PQQ, whereas mutants defective in plant growth promotion do not. Application of wild-type B16 on tomato (Solanum lycopersicum) plants cultivated in a hydroponic culture system significantly increased the height, flower number, fruit number, and total fruit weight, whereas none of the strains that did not produce PQQ promoted tomato growth. Furthermore, 5 to 1,000 nm of synthetic PQQ conferred a significant increase in the fresh weight of cucumber (Cucumis sativus) seedlings, confirming that PQQ is a plant growth promotion factor. Treatment of cucumber leaf discs with PQQ and wild-type B16 resulted in the scavenging of reactive oxygen species and hydrogen peroxide, suggesting that PQQ acts as an antioxidant in plants.

Severe wilt symptoms similar to bacterial wilt caused by Ralstonia solanacearum were observed in tomato, hot pepper, eggplant, potato, perilla, sesame, and sunflower in 2000 and 2001 in Korea. From diseased crops at 65 different locations, we obtained 106 isolates that produced green pigment on CPG medium; 36 were isolated from discolored rice panicles. The causal pathogen was identified as Burkholderia glumae based on its biochemical characteristics, fatty acid methyl ester analysis, and 16S rRNA gene sequence. Nine representative isolates produced toxoflavin, as determined by electrospray ionization mass spectrometry using a direct inlet system and TLC analyses, and caused bacterial wilt on tomato, sesame, perilla, eggplant, and hot pepper. However, BGR12, a wild-type isolate lacking toxoflavin production and toxoflavin-deficient mutants generated by Tn5lacZ failed to cause bacterial wilt on those five field crops. Cells of B. glumae and synthetic toxoflavin caused wilt symptoms on field crops, demonstrating a lack of host specificity. Synthetic toxoflavin caused wilt symptoms on tomato, sesame, perilla, eggplant, and hot pepper at 10 μg/ml concentration 1 day after treatment. This is the first report of bacterial wilt on various crops caused by B. glumae, and our results clearly demonstrate that toxoflavin is a key factor in wilt symptom development.

Summary: The bacterium Burkholderia glumae causes rice grain rot by producing toxoflavin, whose expression is regulated by quorum sensing (QS). We report a major deviation from the current paradigm for the regulation of bacterial polar flagellum genes. The N‐octanoyl homoserine lactone (C8‐HSL)‐deficient mutant of B. glumae is aflagellate and has lost the ability to swim and swarm at 37°C. Mutagenesis of the bacterium with the mini‐Tn5rescue identified an IclR‐type transcriptional regulator, called QsmR, which is important for flagellum formation. TofR, which is a cognate C8‐HSL receptor, activated qsmR expression by binding directly to the qsmR promoter region. From the flagellum gene cluster, we identified flhDC homologues that are directly activated by QsmR. FlhDC in turn activates the expression of genes involved in flagellum biosynthesis, motor functions and chemotaxis in B. glumae. Non‐motile qsmR, fliA and flhDC mutants produced toxoflavin, but lost pathogenicity for rice. The unexpected discovery of FlhDC in a polarly flagellate bacterium suggests that exceptions to the typical regulatory mechanisms of flagellum genes exist in Gram‐negative bacteria. The finding that functional flagella play critical roles in the pathogenicity of B. glumae suggests that either QS or flagellum formation constitutes a good target for the control of rice grain rot.

Summary: Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a unipolar polysaccharide (UPP) adhesin at single cell poles that contact surfaces. Here we report that elevated levels of the intracellular signal cyclic diguanosine monophosphate (c‐di‐GMP) lead to surface‐contact‐independent UPP production and a red colony phenotype due to production of UPP and the exopolysaccharide cellulose, when A. tumefaciens is incubated with the polysaccharide stain Congo Red. Transposon mutations with elevated Congo Red staining identified presumptive UPP‐negative regulators, mutants for which were hyperadherent, producing UPP irrespective of surface contact. Multiple independent mutations were obtained in visN and visR, activators of flagellar motility in A. tumefaciens, now found to inhibit UPP and cellulose production. Expression analysis in a visR mutant and isolation of suppressor mutations, identified three diguanylate cyclases inhibited by VisR. Null mutations for two of these genes decrease attachment and UPP production, but do not alter cellular c‐di‐GMP levels. However, analysis of catalytic site mutants revealed their GGDEF motifs are required to increase UPP production and surface attachment. Mutations in a specific presumptive c‐di‐GMP phosphodiesterase also elevate UPP production and attachment, consistent with c‐di‐GMP activation of surface‐dependent adhesin deployment.