Oliver Kirchner

Insights into Genome Plasticity and Pathogenicity of the Plant Pathogenic Bacterium Xanthomonas campestris pv. vesicatoria Revealed by the Complete Genome Sequence

The gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causative agent of bacterial spot disease in pepper and tomato plants, which leads to economically important yield losses. This pathosystem has become a well-established model for studying bacterial infection strategies. Here, we present the whole-genome sequence of the pepper-pathogenic Xanthomonas campestris pv. vesicatoria strain 85-10, which comprises a 5.17-Mb circular chromosome and four plasmids. The genome has a high G+C content (64.75%) and signatures of extensive genome plasticity. Whole-genome comparisons revealed a gene order similar to both Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and a structure completely different from Xanthomonas oryzae pv. oryzae. A total of 548 coding sequences (12.2%) are unique to X. campestris pv. vesicatoria. In addition to a type III secretion system, which is essential for pathogenicity, the genome of strain 85-10 encodes all other types of protein secretion systems described so far in gram-negative bacteria. Remarkably, one of the putative type IV secretion systems encoded on the largest plasmid is similar to the Icm/Dot systems of the human pathogens Legionella pneumophila and Coxiella burnetii. Comparisons with other completely sequenced plant pathogens predicted six novel type III effector proteins and several other virulence factors, including adhesins, cell wall-degrading enzymes, and extracellular polysaccharides.

Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum.

During the last decades, the gram-positive soil bacterium Corynebacterium glutamicum has been shown to be a very versatile microorganism for the large-scale fermentative production of L-amino acids. Up to now, a vast amount of techniques and tools for genetic engineering and amplification of relevant structural genes have been developed. The objectives of this study are to summarize the published literature on tools for genetic engineering in C. glutamicum and to focus on new sophisticated and highly efficient methods in the fields of DNA transfer techniques, cloning vectors, integrative genetic tools, and antibiotic-free self-cloning. This repertoire of C. glutamicum methodology provides an experimental basis for efficient genetic analyses of the recently completed genome sequence.

Efficient Electrotransformation of Corynebacterium diphtheriae with a Mini-Replicon Derived from the Corynebacterium glutamicum Plasmid pGA1

Efficient transformation of the human pathogen Corynebacterium diphtheriae was achieved with novel cloning vectors consisting of a mini-replicon from the cryptic C. glutamicum plasmid pGA1 as well as of the aph(3′)-IIa or tetA(Z) antibiotic resistance genes. Plasmid-containing transformants of C. diphtheriae were recovered at frequencies ranging from 1.3 × 105 to 4.8 × 106 colony forming units (cfu)/μg of plasmid DNA. Vector DNA was directly transferred from Escherichia coli into C. diphtheriae with frequencies up to 5.6 × 105 cfu/μg of plasmid DNA. On the basis of the pGA1 mini-replicon, an expression vector system was established for C. diphtheriae by means of the Ptac promoter and the green fluorescent reporter protein. In addition, other commonly used vector systems from C. glutamicum, including the pBL1 and pHM1519 replicons, and the sacB conditionally lethal selection marker from Bacillus subtilis, were shown to be functional in C. diphtheriae. Thus, the ability to apply the standard methods of C. glutamicum recombinant DNA technology will greatly facilitate the functional analysis of the recently completed C. diphtheriae genome sequence.

The genome sequence of the tomato-pathogenic actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382 reveals a large island involved in pathogenicity

Clavibacter michiganensis subsp. michiganensis is a plant-pathogenic actinomycete that causes bacterial wilt and canker of tomato. The nucleotide sequence of the genome of strain NCPPB382 was determined. The chromosome is circular, consists of 3.298 Mb, and has a high G+C content (72.6%). Annotation revealed 3,080 putative protein-encoding sequences; only 26 pseudogenes were detected. Two rrn operons, 45 tRNAs, and three small stable RNA genes were found. The two circular plasmids, pCM1 (27.4 kbp) and pCM2 (70.0 kbp), which carry pathogenicity genes and thus are essential for virulence, have lower G+C contents (66.5 and 67.6%, respectively). In contrast to the genome of the closely related organism Clavibacter michiganensis subsp. sepedonicus, the genome of C. michiganensis subsp. michiganensis lacks complete insertion elements and transposons. The 129-kb chp/tomA region with a low G+C content near the chromosomal origin of replication was shown to be necessary for pathogenicity. This region contains numerous genes encoding proteins involved in uptake and metabolism of sugars and several serine proteases. There is evidence that single genes located in this region, especially genes encoding serine proteases, are required for efficient colonization of the host. Although C. michiganensis subsp. michiganensis grows mainly in the xylem of tomato plants, no evidence for pronounced genome reduction was found. C. michiganensis subsp. michiganensis seems to have as many transporters and regulators as typical soil-inhabiting bacteria. However, the apparent lack of a sulfate reduction pathway, which makes C. michiganensis subsp. michiganensis dependent on reduced sulfur compounds for growth, is probably the reason for the poor survival of C. michiganensis subsp. michiganensis in soil.

New type III effectors from Xanthomonas campestris pv. vesicatoria trigger plant reactions dependent on a conserved N-myristoylation motif

Pathogenicity of the gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria depends on a type III secretion system, which translocates bacterial effector proteins into the plant cell. In this study, we identified two novel type III effectors, XopE1 and XopE2 (Xanthomonas outer proteins), using the AvrBs3 effector domain as reporter. XopE1 and XopE2 belong to the HopX family and possess a conserved putative N-myristoylation motif that is also present in the effector XopJ from X. campestris pv. vesicatoria 85-10. XopJ is a member of the YopJ/AvrRxv family of acetyltransferases. Confocal laser scanning microscopy and immunocytochemistry revealed that green fluorescent protein fusions of XopE1, XopE2, and XopJ localized to the plant cell plasma membrane. Targeting to the membrane is probably due to N-myristoylation, because a point mutation in the putative myristoylated glycine residue G2 in XopE1, XopE2, and XopJ resulted in cytoplasmic localization of the mutant proteins. Results of hydroxylamine treatments of XopE2 protein extracts suggest that the proteins are additionally anchored in the host cell plasma membrane by palmitoylation. The membrane localization of the effectors strongly influences the phenotypes they trigger in the plant. Agrobacterium-mediated expression of xopE1 and xopJ in Nicotiana benthamiana led to cell-death reactions that, for xopJ, were dependent on the N-myristoylation motif. In the case of xopE1(G2A), cell death was more pronounced with the mutant than with the wild-type protein. In addition, XopE2 has an avirulence activity in Solanum pseudocapsicum.

HpaA from Xanthomonas is a regulator of type III secretion

The Gram‐negative plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to inject effector proteins into the host cell cytoplasm. Efficient secretion of several effector proteins depends on the cytoplasmic global T3S chaperone HpaB. In this study, we show that HpaB interacts with the virulence factor HpaA, which is secreted by the T3S system and translocated into the plant cell. HpaA promotes secretion of pilus, translocon and effector proteins and therefore appears to be an important control protein of the T3S system. Protein–protein interaction studies and the analysis of HpaA deletion derivatives revealed that the C‐terminal protein region, which contains a HpaB binding site, is crucial for the contribution of HpaA to T3S. Secretion of pilus and translocon proteins is not affected when HpaA is expressed as an N‐terminal deletion derivative that lacks the secretion and translocation signal. Our data suggest that binding of HpaA to HpaB within the bacterial cell favours secretion of extracellular components of the secretion apparatus. Secretion of HpaA presumably liberates HpaB and thus promotes effector protein secretion after assembly of the T3S apparatus.

A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp michiganensis

A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 x 10(6) transformants per microg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 x 10(3) transposon mutants per pg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.