Complete genome sequence of the plant pathogen Erwinia amylovora strain ATCC 49946.

Erwinia amylovora causes the economically important disease fire blight that affects rosaceous plants, especially pear and apple. Here we report the complete genome sequence and annotation of strain ATCC 49946. The analysis of the sequence and its comparison with sequenced genomes of closely related enterobacteria revealed signs of pathoadaptation to rosaceous hosts.

Novel quorum-sensing-controlled genes in Erwinia carotovora subsp. carotovora: identification of a fungal elicitor homologue in a soft-rotting bacterium.

Seven new genes controlled by the quorum-sensing signal molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) have been identified in Erwinia carotovora subsp. carotovora. Using TnphoA as a mutagen, we enriched for mutants defective in proteins that could play a role in the interaction between E. carotovora subsp. carotovora and its plant hosts, and identified NipEcc and its counterpart in E. carotovora subsp. atroseptica. These are members of a growing family of proteins related to Nep1 from Fusarium oxysporum which can induce necrotic responses in a variety of dicotyledonous plants. NipEcc produced necrosis in tobacco, NipEca affected potato stem rot, and both affected virulence in potato tubers. In E. carotovora subsp. carotovora, nip was shown to be subject to weak repression by the LuxR family regulator, EccR, and may be regulated by the negative global regulator RsmA.

Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors.

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.

Genome sequence of Yersinia pestis, the causative agent of plague.

The Gram-negative bacterium Yersinia pestis is the causative agent of the systemic invasive infectious disease classically referred to as plague, and has been responsible for three human pandemics: the Justinian plague (sixth to eighth centuries), the Black Death (fourteenth to nineteenth centuries) and modern plague (nineteenth century to the present day). The recent identification of strains resistant to multiple drugs and the potential use of Y. pestis as an agent of biological warfare mean that plague still poses a threat to human health. Here we report the complete genome sequence of Y. pestis strain CO92, consisting of a 4.65-megabase (Mb) chromosome and three plasmids of 96.2 kilobases (kb), 70.3 kb and 9.6 kb. The genome is unusually rich in insertion sequences and displays anomalies in GC base-composition bias, indicating frequent intragenomic recombination. Many genes seem to have been acquired from other bacteria and viruses (including adhesins, secretion systems and insecticidal toxins). The genome contains around 150 pseudogenes, many of which are remnants of a redundant enteropathogenic lifestyle. The evidence of ongoing genome fluidity, expansion and decay suggests Y. pestis is a pathogen that has undergone large-scale genetic flux and provides a unique insight into the ways in which new and highly virulent pathogens evolve.

Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18.

Salmonella enterica serovar Typhi (S. typhi) is the aetiological agent of typhoid fever, a serious invasive bacterial disease of humans with an annual global burden of approximately 16 million cases, leading to 600,000 fatalities. Many S. enterica serovars actively invade the mucosal surface of the intestine but are normally contained in healthy individuals by the local immune defence mechanisms. However, S. typhi has evolved the ability to spread to the deeper tissues of humans, including liver, spleen and bone marrow. Here we have sequenced the 4,809,037-base pair (bp) genome of a S. typhi (CT18) that is resistant to multiple drugs, revealing the presence of hundreds of insertions and deletions compared with the Escherichia coli genome, ranging in size from single genes to large islands. Notably, the genome sequence identifies over two hundred pseudogenes, several corresponding to genes that are known to contribute to virulence in Salmonella typhimurium. This genetic degradation may contribute to the human-restricted host range for S. typhi. CT18 harbours a 218,150-bp multiple-drug-resistance incH1 plasmid (pHCM1), and a 106,516-bp cryptic plasmid (pHCM2), which shows recent common ancestry with a virulence plasmid of Yersinia pestis.

Analysis of the carbapenem gene cluster of Erwinia carotovora: definition of the antibiotic biosynthetic genes and evidence for a novel beta-lactam resistance mechanism.

Members of two genera of Gram-negative bacteria, Serratia and Erwinia, produce a beta-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid. We have reported previously the cloning and sequencing of the genes responsible for production of this carbapenem in Erwinia carotovora. These genes are organized as an operon, carA--H, and are controlled by a LuxR-type transcriptional activator, encoded by the linked carR gene. We report in this paper the genetic dissection of this putative operon to determine the function of each of the genes. We demonstrate by mutational analysis that the products of the first five genes of the operon are involved in the synthesis of the carbapenem molecule. Three of these, carABC, are absolutely required. In addition, we provide evidence for the existence of a novel carbapenem resistance mechanism, encoded by the CarF and carG genes. Both products of these overlapping and potentially translationally coupled genes have functional, N-terminal signal peptides. Removal of these genes from the Erwinia chromosome results in a carbapenem-sensitive phenotype. We assume that these novel beta-lactam resistance genes have evolved in concert with the biosynthetic genes to ensure 'self-resistance' in the Erwinia carbapenem producer.

Analysis of bacterial carbapenem antibiotic production genes reveals a novel beta-lactam biosynthesis pathway.

Carbapenems are beta-lactam antibiotics which have an increasing utility in chemotherapy, particularly for nosocomial, multidrug-resistant infections. Strain GS101 of the bacterial phytopathogen, Erwinia carotovora, makes the simple beta-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid. We have mapped and sequenced the Erwinia genes encoding carbapenem production and have cloned these genes into Escherichia coli where we have reconstituted, for the first time, functional expression of the beta-lactam in a heterologous host. The carbapenem synthesis gene products are unrelated to enzymes involved in the synthesis of the so-called sulphur-containing beta-lactams, namely penicillins, cephamycins and cephalosporins. However, two of the carbapenem biosynthesis genes, carA and carC, encode proteins which show significant homology with proteins encoded by the Streptomyces clavuligerus gene cluster responsible for the production of the beta-lactamase inhibitor, clavulanic acid. These homologies, and some similarities in genetic organization between the clusters, suggest an evolutionary relatedness between some of the genes encoding production of the antibiotic and the beta-lactamase inhibitor. Our observation are consistent with the evolution of a second major biosynthetic route to the production of beta-lactam-ring-containing antibiotics.

Analysis of bacterial carbapenem antibiotic production genes reveals a novel ß-lactam biosynthesis pathway.

Carbapenems are ß-lactam antibiotics which have an increasing utility in chemotherapy, particularly for nosocomial, multidrug-resistant infections. Strain GS101 of the bacterial phytopathogen, Erwinia carotovora, makes the simple ß-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid. We have mapped and sequenced the Erwinia genes encoding carbapenem production and have cloned these genes into Escherichia coli where we have reconstituted, for the first time, functional expression of the ß-lactam in a heterologous host. The carbapenem synthesis gene products are unrelated to enzymes involved in the synthesis of the so-called sulphur-containing ß-lactams, namely penicillins, cephamycins and cephalosporins. However, two of the carbapenem biosynthesis genes, carA and carC, encode proteins which show significant homology with proteins encoded by the Streptomycesclavuligerus gene cluster responsible for the production of the ß-lactamase inhibitor, clavulanic acid. These homologies, and some similarities in genetic organization between the clusters, suggest an evolutionary relatedness between some of the genes encoding production of the antibiotic and the ß-lactamase inhibitor. Our observations are consistent with the evolution of a second major biosynthetic route to the production of ß-lactam-ring-containing antibiotics.

Carbapenem antibiotic production in Erwinia carotovora is regulated by CarR, a homologue of the LuxR transcriptional activator.

Strain GS101 of Erwinia carotovora makes the carbapenem antibiotic, 1-carbapen-2-em-3-carboxylic acid. Mutants defective in antibiotic production can be assigned to two groups, group 1 and group 2. Group 2 mutants are defective in the carl gene encoding a protein responsible for synthesis of the Lux autoinducer N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), which is required to induce carbapenem synthesis in strain GS101. In this paper we describe the molecular genetic analysis of the group 1 mutants which we presumed were defective in the carbapenem biosynthesis (car) genes. We isolated a cosmid (cWU142) that complemented the group 1 mutants of strain GS101. A small (1.03 kb) subclone of cWU142 complemented most of the group 1 mutants, and the sequence revealed that the relevant gene (carR) encodes a homologue of the Vibrio fischeri LuxR protein. A disproportionately high frequency of carR mutants arose in strain GS101 and this was due to carR acting as a 'hot spot' target for secondary transposition of a Tn5 element in this strain. The CarR protein joins a rapidly growing list of homologues, found in taxonomically unrelated bacteria, which act as positive transcriptional activators of genes encoding diverse metabolic functions, including bioluminescence, exoenzyme virulence factor synthesis, cell division, plasmid conjugation, rhizosphere-specific gene induction, surfactant synthesis and antibiotic production. Most of these LuxR-type regulators have been shown to depend, for their function, on N-acyl homoserine lactones, which act as chemical signals enabling co-ordination of gene expression with cell density.

Cloning and sequence analysis of the glucose-6-phosphate dehydrogenase gene from the cyanobacterium Synechococcus PCC 7942.

The glucose-6-phosphate dehydrogenase (EC 1.1.1.49) gene (zwf) of the cyanobacterium Synechococcus PCC 7942 was cloned on a 2.8 kb Hind III fragment. Sequence analysis revealed an ORF of 1572 nucleotides encoding a polypeptide of 524 amino acids which exhibited 41% identity with the glucose-6-phosphate dehydrogenase of Escherichia coli.