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Clavibacter michiganensis ssp. michiganensis (Cmm) causes substantial economic losses in tomato production worldwide. The disease symptoms observed in plants infected systemically by Cmm are wilting and canker on the stem, whereas blister-like spots develop in locally infected leaves. A wide repertoire of serine proteases and cell wall-degrading enzymes has been implicated in the development of wilt and canker symptoms. However, virulence factors involved in the formation of blister-like spots, which play an important role in Cmm secondary spread in tomato nurseries, are largely unknown. Here, we demonstrate that Cmm virulence factors play different roles during blister formation relative to wilting. Inoculation with a green fluorescent protein (GFP)-labelled Cmm382 indicates that penetration occurs mainly through trichomes. When spray inoculated on tomato leaves, the wild-type Cmm382 and Cmm100 (lacking plasmids pCM1 and pCM2) strains form blister-like spots on leaves, whereas Cmm27 (lacking the chp/tomA pathogenicity island) is non-pathogenic, indicating that plasmid-borne genes, which have a crucial role in wilting, are not required for blister formation. Conversely, mutations in chromosomal genes encoding serine proteases (chpC and sbtA), cell wall-degrading enzymes (pgaA and endX/Y), a transcriptional regulator (vatr2), a putative perforin (perF) and a putative sortase (srtA) significantly affect disease incidence and the severity of blister formation. The transcript levels of these genes, as measured by quantitative reverse transcription-polymerase chain reaction, showed that, during blister formation, they are expressed early at 8-16 h after inoculation, whereas, during wilting, they are expressed after 24-72 h or expressed at low levels. Plant gene expression studies suggest that chpC is involved in the suppression of host defence.

Clavibacter michiganensis ssp. michiganensis (Cmm) causes substantial economic losses in tomato production worldwide. The disease symptoms observed in plants infected systemically by Cmm are wilting and canker on the stem, whereas blister‐like spots develop in locally infected leaves. A wide repertoire of serine proteases and cell wall‐degrading enzymes has been implicated in the development of wilt and canker symptoms. However, virulence factors involved in the formation of blister‐like spots, which play an important role in Cmm secondary spread in tomato nurseries, are largely unknown. Here, we demonstrate that Cmm virulence factors play different roles during blister formation relative to wilting. Inoculation with a green fluorescent protein (GFP)‐labelled Cmm382 indicates that penetration occurs mainly through trichomes. When spray inoculated on tomato leaves, the wild‐type Cmm382 and Cmm100 (lacking plasmids pCM1 and pCM2) strains form blister‐like spots on leaves, whereas Cmm27 (lacking the chp/tomA pathogenicity island) is non‐pathogenic, indicating that plasmid‐borne genes, which have a crucial role in wilting, are not required for blister formation. Conversely, mutations in chromosomal genes encoding serine proteases (chpC and sbtA), cell wall‐degrading enzymes (pgaA and endX/Y), a transcriptional regulator (vatr2), a putative perforin (perF) and a putative sortase (srtA) significantly affect disease incidence and the severity of blister formation. The transcript levels of these genes, as measured by quantitative reverse transcription‐polymerase chain reaction, showed that, during blister formation, they are expressed early at 8–16 h after inoculation, whereas, during wilting, they are expressed after 24–72 h or expressed at low levels. Plant gene expression studies suggest that chpC is involved in the suppression of host defence.

Bacteriophage CMP1 is a member of the Siphoviridae family that infects specifically the plant-pathogen Clavibacter michiganensis subsp. michiganensis. The linear double- stranded DNA is terminally redundant and not circularly permuted. The complete nucleotide sequence of the bacteriophage CMP1 genome consists of 58,652 bp including the terminal redundant ends of 791 bp. The G+C content of the phage (57%) is significantly lower than that of its host (72.66%). 74 potential open reading frames were identified and annotated by different bioinformatic tools. Two large clusters which encode the early and the late functions could be identified which are divergently transcribed. There are only a few hypothetical gene products with conserved domains and significant similarity to sequences from the databases. Functional analyses confirmed the activity of four gene products, an endonuclease, an exonuclease, a single-stranded DNA binding protein and a thymidylate synthase. Partial genomic sequences of CN77, a phage of Clavibacter michiganensis subsp. nebraskensis, revealed a similar genome structure and significant similarities on the level of deduced amino acid sequences. An endolysin with peptidase activity has been identified for both phages, which may be good tools for disease control of tomato plants against Clavibacter infections.

To detect plasmid-borne antibiotic-resistance genes in wastewater treatment plant (WWTP) bacteria, 192 resistance-gene-specific PCR primer pairs were designed and synthesized. Subsequent PCR analyses on total plasmid DNA preparations obtained from bacteria of activated sludge or the WWTP's final effluents led to the identification of, respectively, 140 and 123 different resistance-gene-specific amplicons. The genes detected included aminoglycoside, β-lactam, chloramphenicol, fluoroquinolone, macrolide, rifampicin, tetracycline, trimethoprim and sulfonamide resistance genes as well as multidrug efflux and small multidrug resistance genes. Some of these genes were only recently described from clinical isolates, demonstrating genetic exchange between clinical and WWTP bacteria. Sequencing of selected resistance-gene-specific amplicons confirmed their identity or revealed that the amplicon nucleotide sequence is very similar to a gene closely related to the reference gene used for primer design. These results demonstrate that WWTP bacteria are a reservoir for various resistance genes. Moreover, detection of about 64 % of the 192 reference resistance genes in bacteria obtained from the WWTP's final effluents indicates that these resistance determinants might be further disseminated in habitats downstream of the sewage plant.

Composition and gene content of a biogas-producing microbial community from a production-scale biogas plant fed with renewable primary products was analysed by means of a metagenomic approach applying the ultrafast 454-pyrosequencing technology. Sequencing of isolated total community DNA on a Genome Sequencer FLX System resulted in 616,072 reads with an average read length of 230 bases accounting for 141,664,289 bases sequence information. Assignment of obtained single reads to COG (Clusters of Orthologous Groups of proteins) categories revealed a genetic profile characteristic for an anaerobic microbial consortium conducting fermentative metabolic pathways. Assembly of single reads resulted in the formation of 8752 contigs larger than 500 bases in size. Contigs longer than 10kb mainly encode house-keeping proteins, e.g. DNA polymerase, recombinase, DNA ligase, sigma factor RpoD and genes involved in sugar and amino acid metabolism. A significant portion of contigs was allocated to the genome sequence of the archaeal methanogen Methanoculleus marisnigri JR1. Mapping of single reads to the M. marisnigri JR1 genome revealed that approximately 64% of the reference genome including methanogenesis gene regions are deeply covered. These results suggest that species related to those of the genus Methanoculleus play a dominant role in methanogenesis in the analysed fermentation sample. Moreover, assignment of numerous contig sequences to clostridial genomes including gene regions for cellulolytic functions indicates that clostridia are important for hydrolysis of cellulosic plant biomass in the biogas fermenter under study. Metagenome sequence data from a biogas-producing microbial community residing in a fermenter of a biogas plant provide the basis for a rational approach to improve the biotechnological process of biogas production.

Corynebacterium kroppenstedtii is a lipophilic corynebacterial species that lacks in the cell envelope the characteristic alpha-alkyl-beta-hyclroxy long-chain fatty acids, designated mycolic acids. We report here the bioinformatic analysis of genome data obtained by pyrosequencing of the type strain C kroppenstedtii DSM44385 that was initially isolated from human Sputum. A single run with the Genome Sequencer FLX system revealed 560,248 Shotgun reads with 110,018,974 detected bases that were assembled into a contiguous genomic sequence with a total size of 2,446,804 bp. Automatic annotation of the complete genome sequence resulted in the prediction of 2122 coding sequences, of which 29% were considered as specific for C. kroppenstedtii when compared with predicted proteins from hitherto sequenced pathogenic corynebacteria. This Comparative content analysis of the genome data revealed a large repertoire of genes involved in sugar Uptake and central carbohydrate metabolism and the presence of the mevalonate route for isoprenoid biosynthesis. The lack of mycolic acids and the lipophilic lifestyle of C. kroppenstedtii are apparently caused by gene loss, including a condensase gene cluster, a mycolate reductase gene, and a microbial type I fatty acid synthase gene. A complete beta-oxidation pathway involved in the degradation of fatty acids is present in the genome. Evaluation of the genomic data indicated that lipophilism is the dominant feature involved in pathogenicity of C. kroppenstedtii. (C) 2008 Elsevier B.V. All rights reserved.

A total community DNA sample from an agricultural biogas reactor continuously fed with maize silage, green rye, and small proportions of chicken manure has recently been sequenced using massively parallel pyrosequencing. In this Study, the sample was computationally characterized without a prior assembly step, providing quantitative insights into the taxonomic composition and gene content of the underlying microbial community. Clostridiales from the phylum Firmicutes is the most prevalent phylogenetic order, Methanomicrobiales are dominant among methanogenic archaea. An analysis of Operational Taxonomic Units (OTUs) revealed that the entire microbial community is only partially covered by the sequenced sample, despite that estimates suggest only a moderate overall diversity of the community. Furthermore, the results strongly indicate that archaea related to the genus Methanoculleus, using CO2 as electron acceptor and H-2 as electron donor, are the main producers of methane in the analyzed biogas reactor sample. A phylogenetic analysis of glycosyl hydrolase protein families suggests that Clostridia play an important role in the digestion of polysaccharides and oligosaccharides. Finally, the results unveiled that most of the organisms constituting the sample are still unexplored. (C) 2008 Elsevier B.V. All rights reserved.

The complete nucleotide sequence of plasmid pA387 of Amycolatopsis benzoatilytica DSM 43387 was determined. Sequence analysis revealed that pA387 is 30,157 bp long and has a G+C content of 71.74%. To obtain a minimal transferable replicon capable of self-replication, a 2,176 bp fragment of pA387 was cloned, and we demonstrated that this fragment is sufficient for autonomous replication. The replication region of pA387 exhibited no significant homology to any known replication proteins available in databases. Putative maintenance and transfer functions were identified on pA387. The predicted products of open reading frames, ORF 2 and ORF 12, resembled the plasmid stabilizing proteins, a DNA resolvase and a ParA protein, respectively. The putative translational products of ORF 15 and ORF 16 showed similarity to known bacterial conjugation proteins, TraG and TraA, respectively. A conjugative Escherichia coli -Amycolatopsis shuttle-cloning vector was constructed by using the pA387 replicon and designated pSETRL1. Shuttle vector pSETRL1 successfully transformed Amycolatopsis mediterranei DSM 40773 and Amycolatopsis orientalis NBRC 12806 by conjugation and electroporation, and is likely to be a useful vector in Amycolatopsis research. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The plant pathogen Clavibacter michiganensis subsp. michiganensis is a gram-positive bacterium responsible for wilt and canker disease of tomato. Although disease development is well characterized and diagnosed, molecular mechanisms of C. michiganensis subsp. michiganensis virulence are poorly understood. Here, we identified and characterized two C. michiganensis subsp. michiganensis transcriptional regulators, Vatr1 and Vatr2, that are involved in pathogenicity of C. michiganensis subsp. michiganensis. Vatr1 and Vatr2 belong to TetR and MocR families of transcriptional regulators, respectively. Mutations in their corresponding genes caused attenuated virulence, with the Δvatr2 mutant showing a more dramatic effect than Δvatr1. Although both mutants grew well in vitro and reached a high titer in planta, they caused reduced wilting and canker development in infected plants compared with the wild-type bacterium. They also led to a reduced expression of the ethylene-synthesizing tomato enzyme ACC-oxidase compared with wild-type C. michiganensis subsp. michiganensis and to reduced ethylene production in the plant. Transcriptomic analysis of wild-type C. michiganensis subsp. michiganensis and the two mutants under infection-mimicking conditions revealed that Vatr1 and Vatr2 regulate expression of virulence factors, membrane and secreted proteins, and signal-transducing proteins. A 70% overlap between the sets of genes positively regulated by Vatr1 and Vatr2 suggests that these transcriptional regulators are on the same molecular pathway responsible for C. michiganensis subsp. michiganensis virulence.

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 × 106 transformants per μg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 × 103 transposon mutants per μg 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.

Chalupowicz, L., Zellermann, E.-M., Fluegel, M., Dror, O., Eichenlaub, R., Gartemann, K.-H., Savidor, A., Sessa, G., Iraki, N., Barash, I., and Manulis-Sasson, S. 2012. Colonization and movement of GFP-labeled Clavibacter michiganensis subsp. michiganensis during tomato infection. Phytopathology 102:23-31. The vascular pathogen Clavibacter michiganensis subsp. michiganensis is responsible for bacterial wilt and canker of tomato. Pathogenicity of this bacterium is dependent on plasmid-borne virulence factors and serine proteases located on the chromosomal chp/tomA pathogenicity island (PAI). In this study, colonization patterns and movement of C.;michiganensis subsp. michiganensis during tomato infection was examined using a green fluorescent protein (GFP)-labeled strain. A plasmid expressing GFP in C. michiganensis subsp. michiganensis was constructed and found to be stable in planta for at least 1 month. Confocal laser-scanning microscopy (CLSM) of inoculated stems showed that the pathogen extensively colonizes the lumen of xylem vessels and preferentially attaches to spiral secondary wall thickening of the protoxylem. Acropetal movement of the wild-type strain C. michiganensis subsp. michiganensis NCPPB382 (Cmm382) in tomato resulted in an extensive systemic colonization of the whole plant reaching the apical region after 15 days, whereas Cmm100 (lacking the plasmids pCM1 and pCM2) or Cmm27 (lacking the chp/tomA PAI) remained confined to the area surrounding of the inoculation site. Cmm382 formed biofilm-like structures composed of large bacterial aggregates on the interior of xylem walls as observed by CLSM and scanning electron microscopy. These findings suggest that virulence factors located on the chp/tomA PAI or the plasmids are required for effective movement of the pathogen in tomato and for the formation of cellular aggregates.

Sixty-eight Clavibacter michiganensis subsp. michiganensis (Cmm) strains from recent outbreaks of bacterial wilt and canker in Serbia were collected from several tomato growing regions during a three-year period. The pathogen was identified based on bacteriological characteristics and pathogenicity tests and the identity of strains was confirmed by DAS ELISA and PCR amplification using primers CMM5/6 and PSA4/R. The strains showed homogeneity in biochemical and physiological properties. However, pathogenicity tests revealed differences in virulence that are presumably due to a loss of the pat-1 gene. Further strain characterization using DNA-based methods revealed a high diversity of the Serbian Cmm strains. Based on multi-locus sequence typing (MLST) analyses of five genes, Cmm strains were divided into seven groups. The pulsed-field gel electrophoresis (PFGE) pattern of a selection of strains supported the groupings based on trees of the kdpA/sdhA sequences. On the other hand, groupings made according to PFGE and MLST were not correlated to plasmid content in all cases. This study suggested that high genetic variability of the Serbian Cmm strains was detected both in MLST and PFGE analyses, and could have resulted either from new Cmm strains being introduced by seeds from different origins or as a consequence of an intraspecific hybridization process. In addition, this study proposed MLST as an efficient tool in epidemiological studies, population biology investigations and tracking the routes of transmission of pathogens. Four of the five house-keeping genes (kdpA, sdhA, ligA and gyrB) selected to characterize Cmm strains proved to be suitable for the MLST analysis. This is the first study carried out on the characterization of Cmm using MLST.

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) causes wilt and canker disease of tomato (Solanum lycopersicum). Mechanisms of Cmm pathogenicity and tomato response to Cmm infection are not well understood. To explore the interaction between Cmm and tomato, multidimensional protein identification technology (MudPIT) and tandem mass spectrometry were used to analyze in vitro and in planta generated samples. The results show that during infection Cmm senses the plant environment, transmits signals, induces, and then secretes multiple hydrolytic enzymes, including serine proteases of the Pat-1, Ppa, and Sbt familes, the CelA, XysA, and NagA glycosyl hydrolases, and other cell wall-degrading enzymes. Tomato induction of pathogenesis-related (PR) proteins, LOX1, and other defense-related proteins during infection indicates that the plant senses the invading bacterium and mounts a basal defense response, although partial with some suppressed components including class III peroxidases and a secreted serine peptidase. The tomato ethylene-synthesizing enzyme ACC-oxidase was induced during infection with the wild-type Cmm but not during infection with an endophytic Cmm strain, identifying Cmm-triggered host synthesis of ethylene as an important factor in disease symptom development. The proteomic data were also used to improve Cmm genome annotation, and thousands of Cmm gene models were confirmed or expanded.

Genome-wide expression profiles of the phytopathogenic actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm) strain NCPPB382 were analyzed using a 70mer oligonucleotide microarray. Cmm causes bacterial wilt and canker of tomato, a systemic disease leading to substantial economic losses worldwide. Global gene expression was monitored in vitro after long-and short-term incubation with tomato homogenate to simulate conditions in planta and in vivo ten days after inoculation of tomatoes. Surprisingly, both in the presence of tomato homogenate and in planta known virulence genes (celA, chpC, ppaA/C) were down-regulated indicating that the encoded extracellular enzymes are dispensable in late infection stages where plant tissue has already been heavily destroyed. In contrast, some genes of the tomA-region which are involved in sugar metabolism showed an enhanced RNA-level after permanent growth in supplemented medium. Therefore, these genes may be important for utilization of plant derived nutrients. In the plant Cmm exhibited an expression profile completely different from that in vitro. Especially, the strong expression of genes of the wco-cluster (extracellular polysaccharide II), 10 genes encoding surface or pilus assembly proteins, and CMM_2382, coding for a putative perforin suggest a possible role of these genes in the plant-pathogenic interaction. (C) 2012 Elsevier B.V. All rights reserved.

Clavibacter michiganensis subspecies are actinomycete plant pathogens residing mainly in the xylem vessels that infect economically important host plants. In the Clavibacter subspecies michiganensis and sepedonicus, infecting tomato and potato, respectively, essential factors for disease induction are plasmid encoded and loss of the virulence plasmids converts these biotrophic pathogens into endophytes. The genes responsible for successful colonization of the host plant, including evasion/suppression of plant defense reactions, are chromosomally encoded. Several serine proteases seem to be involved in colonization. They are secreted by Clavibacter, but their targets remain unknown. A type 3 secretion system (T3SS) translocating effectors into the plant cells is absent in these gram-positive pathogens. With the development of the modern ‘omics technologies for RNA and proteins based on the known genome sequences, a new phase in the investigation of the mechanisms of plant pathogenicity has begun to allow the genome-wide investigation of the Clavibacter-host interaction.

Biogas production from renewable resources is attracting increased attention as an alternative energy source due to the limited availability of traditional fossil fuels. Many countries are promoting the use of alternative energy sources for sustainable energy production. In this study, a metagenome from a production-scale biogas fermenter was analysed employing Roche's GS FLX Titanium technology and compared to a previous dataset obtained from the same community DNA sample that was sequenced on the GS FLX platform. Taxonomic profiling based on 16S rRNA-specific sequences and an Environmental Gene Tag (EGT) analysis employing CARMA demonstrated that both approaches benefit from the longer read lengths obtained on the Titanium platform. Results confirmed Clostridia as the most prevalent taxonomic class, whereas species of the order Methanomicrobiales are dominant among methanogenic Archaea. However, the analyses also identified additional taxa that were missed by the previous study, including members of the genera Streptococcus, Acetivibrio, Garciella, Tissierella, and Gelria, which might also play a role in the fermentation process leading to the formation of methane. Taking advantage of the CARMA feature to correlate taxonomic information of sequences with their assigned functions, it appeared that Firmicutes, followed by Bacteroidetes and Proteobacteria, dominate within the functional context of polysaccharide degradation whereas Methanomicrobiales represent the most abundant taxonomic group responsible for methane production. Clostridia is the most important class involved in the reductive CoA pathway (Wood-Ljungdahl pathway) that is characteristic for acetogenesis. Based on binning of 16S rRNA-specific sequences allocated to the dominant genus Methanoculleus, it could be shown that this genus is represented by several different species. Phylogenetic analysis of these sequences placed them in close proximity to the hydrogenotrophic methanogen Methanoculleus bourgensis. While rarefaction analyses still indicate incomplete coverage, examination of the GS FLX Titanium dataset resulted in the identification of additional genera and functional elements, providing a far more complete coverage of the community involved in anaerobic fermentative pathways leading to methane formation.

Background Corynebacterium aurimucosum is a slightly yellowish, non-lipophilic, facultative anaerobic member of the genus Corynebacterium and predominantly isolated from human clinical specimens. Unusual black-pigmented variants of C. aurimucosum (originally named as C. nigricans) continue to be recovered from the female urogenital tract and they are associated with complications during pregnancy. C. aurimucosum ATCC 700975 (C. nigricans CN-1) was originally isolated from a vaginal swab of a 34-year-old woman who experienced a spontaneous abortion during month six of pregnancy. For a better understanding of the physiology and lifestyle of this potential urogenital pathogen, the complete genome sequence of C. aurimucosum ATCC 700975 was determined. Results Sequencing and assembly of the C. aurimucosum ATCC 700975 genome yielded a circular chromosome of 2,790,189 bp in size and the 29,037-bp plasmid pET44827. Specific gene sets associated with the central metabolism of C. aurimucosum apparently provide enhanced metabolic flexibility and adaptability in aerobic, anaerobic and low-pH environments, including gene clusters for the uptake and degradation of aromatic amines, L-histidine and L-tartrate as well as a gene region for the formation of selenocysteine and its incorporation into formate dehydrogenase. Plasmid pET44827 codes for a non-ribosomal peptide synthetase that plays the pivotal role in the synthesis of the characteristic black pigment of C. aurimucosum ATCC 700975. Conclusions The data obtained by the genome project suggest that C. aurimucosum could be both a resident of the human gut and possibly a pathogen in the female genital tract causing complications during pregnancy. Since hitherto all black-pigmented C. aurimucosum strains have been recovered from female genital source, biosynthesis of the pigment is apparently required for colonization by protecting the bacterial cells against the high hydrogen peroxide concentration in the vaginal environment. The location of the corresponding genes on plasmid pET44827 explains why black-pigmented (formerly C. nigricans) and non-pigmented C. aurimucosum strains were isolated from clinical specimens.

Clavibacter michiganensis subsp. michiganensis is a Gram-positive bacterium that causes wilting and cankers, leading to severe economic losses in commercial tomato production worldwide. The disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning, and harvesting. Because of the lack of tools for genetic manipulation, very little is known regarding the mechanisms of seed and seedling infection and movement of C. michiganensis subsp. michiganensis in grafted plants, two focal points for application of bacterial canker control measures in tomato. To facilitate studies on the C. michiganensis subsp. michiganensis movement in tomato seed and grafted plants, we isolated a bioluminescent C. michiganensis subsp. michiganensis strain using the modified Tn 1409 containing a promoterless lux reporter. A total of 19 bioluminescent C. michiganensis subsp. michiganensis mutants were obtained. All mutants tested induced a hypersensitive response in Mirabilis jalapa and caused wilting of tomato plants. Real-time colonization studies of germinating seeds using a virulent, stable, constitutively bioluminescent strain, BL-Cmm17, showed that C. michiganensis subsp. michiganensis aggregated on hypocotyls and cotyledons at an early stage of germination. In grafted seedlings in which either the rootstock or scion was exposed to BL-Cmm17 via a contaminated grafting knife, bacteria were translocated in both directions from the graft union at higher inoculum doses. These results emphasize the use of bioluminescent C. michiganensis subsp. michiganensis to help better elucidate the C. michiganensis subsp. michiganensis -tomato plant interactions. Further, we demonstrated the broader applicability of this tool by successful transformation of C. michiganensis subsp. nebraskensis with Tn 1409 :: lux . Thus, our approach would be highly useful to understand the pathogenesis of diseases caused by other subspecies of the agriculturally important C. michiganensis.

Strains of Arthrobacter catalyze a hydrolytic dehalogenation of 4-chlorobenzoate (4-CBA) to p-hydroxybenzoate. The reaction requires ATP and coenzyme A (CoA), indicating activation of the substrate via a thioester, like that reported for Pseudomonas sp. strain CBS3 (J. D. Scholten, K.-H. Chang, P. C. Babbit, H. Charest, M. Sylvestre, and D. Dunaway-Mariano, Science 253:182-185, 1991). The dehalogenase genes of Arthrobacter sp. strain SU were cloned and expressed in Escherichia coli. Analyses of deletions indicate that dehalogenation depends on three open reading frames (ORFs) which are organized in an operon. There is extensive sequence homology to corresponding gene products in Pseudomonas sp. strain CBS3, suggesting that ORF1 and ORF2 encode a 4-CBA-CoA-ligase and a 4-CBA-CoA dehalogenase, respectively. ORF3 possibly represents a thioesterase, although no homology to the enzyme from Pseudomonas sp. strain CBS3 exists.

Genes for seven putative serine proteases (ChpA-ChpG) belonging to the trypsin subfamily and homologous to the virulence factor pat-1 were identified on the chromosome of Clavibacter michiganensis subsp. michiganensis (Cmm) NCPPB382. All proteases have signal peptides indicating export of these proteins. Their putative function is suggested by two motifs and an aspartate residue typical for serine proteases. Furthermore, six cysteine residues are located at conserved positions. The genes are clustered in a chromosomal region of about 50 kb with a significantly lower G + C content than common for Cmm. The genes chpA, chpB and chpD are pseudogenes as they contain frame shifts and/or in-frame stop codons. The genes chpC and chpG were inactivated by the insertion of an antibiotic resistance cassette. The chpG mutant was not impaired in virulence. However, in planta the titre of the chpC mutant was drastically reduced and only weak disease symptoms were observed. Complementation of the chpC mutant by the wild-type allele restored full virulence. ChpC is the first chromosomal gene of Cmm identified so far that affects the interaction of the pathogen with the host plant.

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.

Clavibacter michiganensis subsp. michiganensis (Cmm) strains, collected during the last decade from different locations in Israel, were analyzed by macrorestriction pulsed-field gel electrophoresis (PFGE). Fifty-eight strains from Israel and 18 from other sources were differentiated into 11 haplotypes with either VspI or DraI restriction enzymes. The strains from Israel formed four distinct groups among which groups A (16 strains) and B (32 strains) constituted the major clusters. These two groups originated from the Besor region, which is the main area for growing tomatoes under cover. Rep-PCR, with either ERIC or BOX primers, confirmed results obtained by PFGE. PCR with primers based on three genes – ppaA, chpC and tomA – that spanned the pathogenicity island of the reference strain NCPPB382, produced the expected products with the tested pathogenic strains. Plasmid analysis of representative strains revealed different profiles of one or two plasmids. However all the strains, including five non-pathogenic ones, reacted positively in PCR with primers based on celA gene, which resides on the plasmid pCM1 of NCPPB382. Southern hybridization of total DNA with a 3.2-kb BglII-fragment of pCM1 containing the celA gene was positive when carried out with 31 strains, but the size of the reacting band was not always the same as that of pCM1, suggesting that the plasmids carrying celA may differ in size. Comparison between the colonization rates of strain Cmm42 (group A) and of Cmm32 (group B) did not show any significant differences. The high diversity of the Cmm strains, on the one hand, and the presence of two persistent groups in the Besor region, on the other hand, suggests that the primary inoculum originated each year from residual plants in the soil rather than from infested seeds, in spite of extensive control measures taken by the growers in this area.

Hybridization of Clavibacter michiganensis subsp. michiganensis total DNA against the pathogenicity gene pat-1 indicated the presence of pat-1 homologous nucteotide sequences on the chromosome and on plasmid pCM2. Isolation of the corresponding DNA fragments and nucleotide sequence determination showed that there are three pat-1 homologous genes: chpA (chromosome) and phpA and phpB (plasmid pCM2). The gene products share common characteristics, i.e. a signal sequence for Sec-dependent secretion, a serine protease motif, and six cysteine residues at conserved positions. Gene chpA located on the chromosome is a pseudogene since it contains a translational stop codon after 97 of 280 amino acids. In contrast to pat-1, cloning of the plasmid encoded homologs phpA and phpB into the avirulent plasmid free Cmm strain CMM100 did not result in a virulent phenotype. So far no proteolytic activity could be demonstrated for Pat-1, however, site specific mutagenesis of pat-1 showed that the serine residue in the motif GDSGG is required for the virulent phenotype of pat-1 and thus Pat-1 could be a functional protease. (c) 2005 Elsevier GmbH. All rights reserved.

The insertion site of a transposon mutant of Clavibacter michiganensis subsp. michiganensis NCPPB382 was cloned and found to be located in the gene tomA encoding a member of the glycosyl hydrolase family 10. The intact gene was obtained from a cosmid library of C. michiganensis subsp. michiganensis. The deduced protein TomA (543 amino acids, 58 kDa) contains a predicted signal peptide and two domains, the N-terminal catalytic domain and a C-terminal fibronectin III-like domain. The closest well-characterized relatives of TomA were tomatinases from fungi involved in the detoxification of the tomato saponin α-tomatine which acts as a growth inhibitor. Growth inhibition of C. michiganensis subsp. michiganensis by α-tomatine was stronger in the tomA mutants than in the wild type. Tomatinase activity assayed by deglycosylation of α-tomatine to tomatidine was demonstrated in concentrated culture supernatants of C. michiganensis subsp. michiganensis. No activity was found with the tomA mutants. However, neither the transposon mutant nor a second mutant constructed by gene disruption was affected in virulence on the tomato cv. Moneymaker.

The organization of lin genes and IS 6100 was studied in three strains of Sphingomonas paucimobilis (B90A, Sp+, and UT26) which degraded hexachlorocyclohexane (HCH) isomers but which had been isolated at different geographical locations. DNA-DNA hybridization data revealed that most of the lin genes in these strains were associated with IS 6100 , an insertion sequence classified in the IS 6 family and initially found in Mycobacterium fortuitum. Eleven, six, and five copies of IS 6100 were detected in B90A, Sp+, and UT26, respectively. IS 6100 elements in B90A were sequenced from five, one, and one regions of the genomes of B90A, Sp+, and UT26, respectively, and were found to be identical. DNA-DNA hybridization and DNA sequencing of cosmid clones also revealed that S. paucimobilis B90A contains three and two copies of linX and linA , respectively, compared to only one copy of these genes in strains Sp+ and UT26. Although the copy number and the sequence of the remaining genes of the HCH degradative pathway ( linB , linC , linD , and linE ) were nearly the same in all strains, there were striking differences in the organization of the linA genes as a result of replacement of portions of DNA sequences by IS 6100 , which gave them a strange mosaic configuration. Spontaneous deletion of linD and linE from B90A and of linA from Sp+ occurred and was associated either with deletion of a copy of IS 6100 or changes in IS 6100 profiles. The evidence gathered in this study, coupled with the observation that the G+C contents of the linA genes are lower than that of the remaining DNA sequence of S. paucimobilis , strongly suggests that all these strains acquired the linA gene through horizontal gene transfer mediated by IS 6100 . The association of IS 6100 with the rest of the lin genes further suggests that IS 6100 played a role in shaping the current lin gene organization.

Clavibacter michiganensis subsp. michiganensis is a plant-pathogenic actinomycete. It infects tomato, spreads through the xylem and causes bacterial wilt and canker. The wild-type strain NCPPB382 carries two plasmids, pCM1 and pCM2. The cured plasmid-free derivative CMM100 is still able to colonize tomato, but no disease symptoms develop indicating that all genes required for successful infection, establishment and growth in the plant reside on the chromosome. Both plasmids carry one virulence factor, a gene encoding a cellulase, CelA in case of pCM1 and a putative serine protease Pat-1 on pCM2. These genes can independently convert the non-virulent strain CMM100 into a pathogen causing wilt on tomatoes. Currently, genome projects for Cmm and the closely related potato-pathogen C. michiganensis subsp. sepedonicus have been initiated. The data from the genome project shall give clues on further genes involved in plant-microbe interaction that can be tested experimentally. Especially, identification of genes related to host-specificity through genome comparison of the two subspecies might be possible. (C) 2003 Elsevier B.V. All rights reserved.

During the last century, large quantities of industrially produced 4-chlorobenzoate (4-CBA) or 4-CBA progenitors (herbicides and polychlorinated biphenyl pesticides) (8, 14, 17, 19) have been released into the environment. A wide variety of 4-CBA-degrading bacterial strains, which are capable of growth on 4-CBA as the principal source of carbon, have been isolated from soil (11, 35). In these bacteria, 4-CBA is first converted to 4-hydroxybenzoate (4-HBA), which is then further metabolized via the ortho- or meta-cleavage pathway (11). The 4-CBA dehalogenation pathway of the 4-CBA-degrading bacterium Pseudomonas sp. strain CBS3 (21) was the first to be characterized. The pathway consists of three reaction steps (28) (see Fig. ​Fig.1)1) which are catalyzed by the enzymes 4-CBA-coenzyme A (CoA) ligase, 4-CBA-CoA dehalogenase, and 4-HBA-CoA thioesterase. Each of these enzymes has been isolated, and their kinetic properties have been defined (7). The three 4-CBA dehalogenation pathway genes are organized within an operon that is under the regulatory control of 4-CBA. In some 4-CBA-degrading bacteria, the gene cluster is located within the chromosomal DNA (6, 23, 26), whereas in others it is carried on a plasmid (22, 25). FIG. 1. Illustration of the reaction steps of the bacterial 4-CBA dehalogenation pathway (28). The Arthrobacter sp. strain SU 4-CBA dehalogenation pathway operon was isolated on a 3.6-kb DNA fragment derived from a 120-kb plasmid (27). The DNA fragment was shown to contain six open reading frames, three of which (fcbA, fcbB, and fcbC) are transcribed in the same direction. Truncation mutants lacking fcbA or fcbB were unable to dehalogenate 4-CBA, whereas the truncation mutant devoid of fcbC was 10-fold less active. The fcbA gene encoded a 57-kDa protein that shares 38% sequence identity with the 57-kDa 4-CBA-CoA ligase of the Pseudomonas sp. strain CBS3 4-CBA dehalogenation pathway. The fcbB gene encoded a 30-kDa protein that shares 50% sequence identity with the 30-kDa 4-CBA-CoA dehalogenase of the Pseudomonas sp. strain CBS3 4-CBA dehalogenation pathway. The fcbA and fcbB genes were thus assumed to encode 4-CBA-CoA ligase and 4-CBA-CoA dehalogenase (27). The neighboring gene, fcbC, encoded a 16-kDa protein that did not share a significant level of sequence homology with the known 16-kDa Pseudomonas sp. strain CBS3 4-HBA-CoA thioesterase. The lack of sequence homology between the Pseudomonas thioesterase and the putative Arthrobacter thioesterase, as well as the different gene order observed in the Pseudomonas (dehalogenase-ligase-thioesterase) and Arthrobacter (ligase-dehalogenase-putative thioesterase) operons, made the assignment of the fcbC as the 4-HBA-CoA thioesterase gene uncertain. In the present study, the fcbC gene was subcloned, and its protein product was characterized.

A new insertion element of 1,449 bp with 25-bp perfect terminal repeats, designated IS 1409 , was identified in the chromosome of 4-chlorobenzoate-degrading Arthrobacter sp. strain TM1 NCIB12013. Upon insertion, IS 1409 causes a target duplication of 8 bp. IS 1409 carries only a single open reading frame of 435 codons encoding the transposase TnpA. Both TnpA and the overall organization of IS 1409 are highly similar to those of some related insertion elements of the ISL 3 group (J. Mahillon and M. Chandler, Microbiol. Mol. Biol. Rev. 62:725–774, 1998). IS 1409 was also found in other 4-chlorobenzoate-degrading Arthrobacter strains and Micrococcus luteus . Based on IS 1409 , a series of transposons carrying resistance genes for chloramphenicol and gentamicin were constructed. These transposons were used to demonstrate transposition events in vivo and to mutagenize Arthrobacter sp. strains.

In this communication we report on a genetically engineered bacterium that reacts by light emission to the presence of 4-chlorobenzoic acid. To construct this strain, DNA fragment (1.7 kb) upstream from the 4-chlorobenzoic acid dehalogenase (fcb) operon of Arthrobacter SU was fused to Vibrio fischeri luxCDABE genes. An Escherichia coli strain transformed with a multi-copy plasmid (pASU) bearing this fusion responded to the presence of 4-chlorobenzoic acid and a few closely related compounds by increased luminescence, exhibiting a high specificity but a relatively low sensitivity. While it could be somewhat, improved by manipulating the experimental pH, sensitivity remained too low for real time applicability. Nevertheless, the principle of using dehalogenase promoters as environmental pollution sensor was demonstrated. (C) 1998 Elsevier Science Ltd. All rights reserved.

Improvement of the antibiotic yield of industrial strains is invariably the main target of industry-oriented research. The approaches used in the past were rational selection, extensive mutagenesis, and biochemical screening. These approaches have their limitations, which are likely to be overcome by the judicious application of recombinant DNA techniques. Efficient cloning vectors and transformation systems have now become available even for antibiotic producers that were previously difficult to manipulate genetically. The genes responsible for antibiotic biosynthesis can now be easily isolated and manipulated. In the first half of this review article, the limitations of classical strain improvement programs and the development of recombinant DNA techniques for cloning and analyzing genes responsible for antibiotic biosynthesis are discussed. The second half of this article addresses some of the major achievements, including the development of genetically engineered microbes, especially with reference to beta-lactams, anthracyclines, and rifamycins.

Publisher Summary The biodegradability of PAHs, dibenzo-p-dioxins, dibenzo furans, and PCBs depends on several parameters, like their bioavailability, the number of aromatic cycles (PAHs), and the degree of halogenation. It is known that aerobic bacteria can degrade PAHs, the non halogenated dibenzo-p-dioxin, dibenzo furan, and biphenyl. The enzymes and genes, involved in this aerobic metabolism of xenobiotics, have been characterized and cloned and it has been demonstrated that many of the enzymes are closely related to the well known catabolism of monocyclic aromatic compounds. The degradation of chlorinated monocyclic aromatic compounds, like, CBAs may occur by two different principles: a) attack on the halogenated compound by broad substrate range oxygenases and further metabolism of halogenated catechols by a modified ortho-pathway: The genes of this catabolism have been cloned, and some scientists were successful in constructing genetically engineered bacteria that can degrade chlorinated biphenyls completely. b) dehalogenation of CBAs before ring-cleavage: It is known that some aerobic bacterial strains can dehalogenase CBAs. This dehalogenation can occur reductively, oxidatively, or hydrolytically.