Microbial tryptophan catabolism affects the vector competence of Anopheles

The influence of microbiota on mosquito physiology and vector competence is becoming increasingly clear but our understanding of interactions between microbiota and mosquitoes still remains incomplete. Here we show that gut microbiota of Anopheles stephensi, a competent malaria vector, participates mosquito tryptophan metabolism. Elimination of microbiota by antibiotics treatment leads to the accumulation of tryptophan (Trp) and its metabolites, kynurenine (Kyn), 3‐hydroxykynurenine (3‐HK) and xanthurenic acid (XA). Of these, 3‐HK impairs the structure of peritrophic matrix (PM), thereby promoting Plasmodium berghei infection. Among the major gut microbiota in An. stephensi, Pseudomonas alcaligenes plays a role in catabolizing 3‐HK as revealed by whole genome sequencing and LC‐MS metabolic analysis. The genome of P. alcaligenes encodes kynureninase (KynU) that is responsible for the conversion of 3‐HK to 3‐Hydroxyanthranilic acid (3‐HAA). Mutation of this gene abrogates the ability of P. alcaligenes to metabolize 3‐HK, which in turn abolishes its role on PM protection. Colonization of An. stephensi with KynU mutated P. alcaligenes fails to protect mosquitoes against parasite infection as effectively as those with wild type bacterium. In summary, we identify an unexpected function of gut microbiota in controlling mosquito tryptophan metabolism with the major consequences on vector competence.

A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis ofMycetohabitans(formerlyBurkholderia)rhizoxinicawith the fungusRhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequencedMycetohabitansspp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. Abtl19-13gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15R. microsporusgenes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealedbtlgenes in 14 diverseMycetohabitansisolates. However, banding patterns and available sequences suggest variation, and thebtl19-13phenotype could not be rescued by abtlgene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in theM. rhizoxinica–R. microsporussymbiosis.

Dynamic Regulation of GacA in Type III Secretion, Pectinase Gene Expression, Pellicle Formation, and Pathogenicity of Dickeya dadantii (Erwinia chrysanthemi 3937)

Dickeya dadantii (Erwinia chrysanthemi 3937) secretes exoenzymes, including pectin-degrading enzymes, leading to the loss of structural integrity of plant cell walls. A type III secretion system (T3SS) is essential for full virulence of this bacterium within plant hosts. The GacS/GacA two-component signal transduction system participates in important biological roles in several gram-negative bacteria. In this study, a gacA deletion mutant (Ech137) of D. dadantii was constructed to investigate the effect of this mutation on pathogenesis and other phenotypes. Compared with wild-type D. dadantii, Ech137 had a delayed biofilm-pellicle formation. The production of pectate lyase (Pel), protease, and cellulase was diminished in Ech137 compared with the wild-type cells. Reduced transcription of two endo-Pel genes, pelD and pelL, was found in Ech137 using a green fluorescence protein-based fluorescence-activated cell sorter promoter activity assay. In addition, the transcription of T3SS genes dspE (an effector), hrpA (a structural protein of the T3SS pilus), and hrpN (a T3SS harpin) was reduced in Ech137. A lower amount of rsmB regulatory RNA was found in gacA mutant Ech137 compared with the wild-type bacterium by quantitative reverse-transcription polymerase chain reaction. Compared with wild-type D. dadantii, a lower amount of hrpL mRNA was observed in Ech137 at 12 h grown in medium. Although the role of RsmA, rsmB, and RsmC in D. dadantii is not clear, from the regulatory pathway revealed in E. carotovora, the lower expression of dspE, hrpA, and hrpN in Ech137 may be due to a posttranscriptional regulation of hrpL through the Gac-Rsm regulatory pathway. Consequently, the reduced exoenzyme production and Pel gene expression in the mutant may be partially due to the regulatory role of rsmB-RsmA on exoenzyme expression. Similar to in vitro results, a lower expression of T3SS and pectinase genes of Ech137 also was observed in bacterial cells inoculated into Saintpaulia ionantha leaves, perhaps accounting for the observed reduction in local maceration. Interestingly, compared with the wild-type D. dadantii, although a lower concentration of Ech137 was observed at day 3 and 4 postinoculation, there is no significant difference in bacterial concentration between the wild-type bacterium and Ech137 in the early stage of infection. Finally, the nearly abolished systemic invasion ability of Ech137 suggests that GacA of D. dadantii is essential for the pathogenicity and systemic movement of the bacterium in S. ionantha.

hrp Genes of Erwinia chrysanthemi 3937 Are Important Virulence Factors

We developed improved virulence assays for Erwinia chrysanthemi 3937 on African violet varieties and devised a new method for the construction of precise bacterial gene knockouts. These methods were tested by constructing mutations in genes suspected to be involved with plant interactions. The virulence of the hrpG and hrcC mutant strains (both gene products presumed to be involved in protein secretion) was greatly reduced on leaves of semitolerant African violet varieties. An hrpN mutant strain produced delayed symptoms on African violet leaves and an hrpN Δpel (Δpel = five major pectate lyase genes deleted) double mutant was nonpathogenic. The hrcC and hrpG mutants did not produce a rapid hypersensitive response (HR) in tobacco, unlike the wild-type bacterium, and the hrpN mutant gave a reduced HR. The results, therefore, establish the importance of hrp genes in the virulence of E. chrysanthemi and their ability to elicit HR on nonhosts. The data also suggest that other effector proteins secreted by the Hrp system are required for full virulence and HR elicitation.

Rhodospirillum rubrum Possesses a Variant of the bchP Gene, Encoding Geranylgeranyl-Bacteriopheophytin Reductase

ABSTRACT The bchP gene product of Rhodobacter sphaeroides is responsible for the reduction of the isoprenoid moiety of bacteriochlorophyll (Bchl) from geranylgeraniol (GG) to phytol; here, we show that this enzyme also catalyzes the reduction of the isoprenoid moiety of bacteriopheophytin (Bphe). In contrast, we demonstrate that a newly identified homolog of this gene in Rhodospirillum rubrum encodes an enzyme, GG-Bphe reductase, capable of reducing the isoprenoid moiety of Bphe only. We propose that Rhodospirillum rubrum is a naturally occurring bchP mutant and that an insertion mutation may have been the initial cause of a partial loss of function. Normal BchP function can be restored to Rhodospirillum rubrum, creating a new transconjugant strain possessing Bchl esterified with phytol. We speculate on the requirement of Rhodospirillum rubrum for phytylated Bphe and on a potential link between the absence of LH2 and of phytylated Bchl from the wild-type bacterium. The identification of a second role for the fully functional BchP in catalyzing the synthesis of phytylated Bphe strongly suggests that homologs of this enzyme may be similarly responsible for the synthesis of phytylated pheophytin in organisms possessing photosystem 2. In addition to bchP, other members of a photosynthesis gene cluster were identified in Rhodospirillum rubrum, including a bchG gene, demonstrated to encode a functional Bchl synthetase by complementation of a Rhodobacter sphaeroides mutant.

Pseudomonas cellulosa expresses a single membrane-bound glycoside hydrolase family 51 arabinofuranosidase

In the accompanying paper [Beylot, McKie, Voragen, Doeswijk-Voragen and Gilbert (2001) Biochem. J. 358, 607–614] the chromosome of Pseudomonas cellulosa was shown to contain two genes, abf51A and abf62A, that encode arabinofuranosidases belonging to glycoside hydrolase families 51 and 62, respectively. In this report we show that expression of Abf51A is induced by arabinose and arabinose-containing polysaccharides. Northern-blot analysis showed that abf51A was efficiently transcribed, whereas no transcript derived from abf62A was detected in the presence of arabinose-containing polysaccharides. Zymogram and Western-blot analyses revealed that Abf51A was located on the outer membrane of P. cellulosa. To investigate the importance of Abf51A in the release of arabinose from poly- and oligosaccharides, transposon mutagenesis was used to construct an abf51A-inactive mutant of P. cellulosa (Δabf51A). The mutant did not grow on linear arabinan or sugar beet arabinan, and utilized arabinoxylan much more slowly than the wild-type bacterium. Arabinofuranosidase activity in Δabf51A against aryl-α-arabinofuranosides, arabinan and α1,5-linked arabino-oligosaccharides was approx. 1% of the wild-type bacterium. The mutant bacterium did not exhibit arabinofuranosidase activity against arabinoxylan, supporting the view that abf62A is not expressed in P. cellulosa. These data indicate that P. cellulosa expresses a membrane-bound glycoside hydrolase family 51 arabinofuranosidase that plays a pivotal role in releasing arabinose from polysaccharides and arabino-oligosaccharides.

Two benzaldehyde dehydrogenases in bacterium N.C.I.B. 8250. Distinguishing properties and regulation

Evidence is presented for the existence in bacterium N.C.I.B. 8250 of two inducible NAD+-linked benzaldehyde dehydrogenases. They may be distinguished in crude extracts by their different thermal stabilities at high pH values, benzaldehyde dehydrogenase I being much more heat-stable than benzaldehyde dehydrogenase II. Only benzaldehyde dehydrogenase I is activated by K+ and certain other univalent cations. Gel-filtration experiments indicate that both enzymes have molecular weights of about 180000. Both enzymes are induced by growth on l-mandelate or phenylglyoxylate; only benzaldehyde dehydrogenase I is gratuitously induced by thiophenoxyacetate and only benzaldehyde dehydrogenase II is induced by benzyl alcohol, by benzaldehyde, and by a number of heterocyclic compounds which do not support growth. Mutants have been isolated that lack either benzaldehyde dehydrogenase II or benzyl alcohol dehydrogenase, or both of the enzymes. Results obtained in induction experiments with the wild-type bacterium N.C.I.B. 8250 and with the mutants show that benzaldehyde dehydrogenase II and benzyl alcohol dehydrogenase are co-ordinately regulated. Overall, the results suggest that benzaldehyde dehydrogenase I is associated with the metabolism of l-mandelate whereas benzaldehyde dehydrogenase II is associated with the metabolism of benzyl alcohol.