Nucleotide Sequences and Organization of the Genes for Carotovoricin (Ctv) from Erwinia carotovora Indicate That Ctv Evolved from the Same Ancestor as Salmonella typhi Prophage

Carotovoricin Er (CtvEr), which is produced by a plant soft rot disease causative agent, Erwinia carotovora subsp. carotovora Er, is a high-molecular-weight bacteriocin showing Myoviridae phage-tail-like morphology with contractile sheath and plural tail fibers. We determined the complete nucleotide sequences of CtvEr genes on the E. carotovora Er chromosome and report that CtvEr genes consist of lysis cassette, major and minor structural protein gene clusters. Four promoters were identified. The lysis gene cassette, which is composed of the genes for lysis enzyme and holin, was also identified and characterized. The nucleotide sequences and organization of the genes for CtvCGE, which is produced by E. carotovora strain CGE234-M403 with the morphology similar to CtvEr, were also determined and compared to that of CtvEr, and it was found that CtvCGE is almost identical to CtvEr except for tail fibers which are involved in the killing spectra of both bacteriocins. We also explain that the gene organization and the deduced amino acid sequences of both carotovoricins are very close to those of prophage, which is lysogenized in the chromosome on Salmonella enterica serovar Typhi CT18. These findings strongly suggest that Ctv evolved as a phage tail-like bacteriocin from a common ancestor with Salmonella typhi prophage.     

DNA inversion in the tail fiber gene alters the host range specificity of carotovoricin Er, a phage-tail-like bacteriocin of phytopathogenic Erwinia carotovora subsp. carotovora Er

Carotovoricin Er is a phage-tail-like bacteriocin produced by Erwinia carotovora subsp. carotovora strain Er, a causative agent for soft rot disease in plants. Here we studied binding and killing spectra of carotovoricin Er preparations for various strains of the bacterium (strains 645Ar, EC-2, N786, and P7) and found that the preparations contain two types of carotovoricin Er with different host specificities; carotovoricin Era possessing a tail fiber protein of 68 kDa killed strains 645Ar and EC-2, while carotovoricin Erb with a tail fiber protein of 76 kDa killed strains N786 and P7. The tail fiber proteins of 68 and 76 kDa had identical N-terminal amino acid sequences for at least 11 residues. A search of the carotovoricin Er region in the chromosome of strain Er indicated the occurrence of a DNA inversion system for the tail fiber protein consisting of (i) two 26-bp inverted repeats inside and downstream of the tail fiber gene that flank a 790-bp fragment and (ii) a putative DNA invertase gene with a 90-bp recombinational enhancer sequence. In fact, when a 1,400-bp region containing the 790-bp fragment was amplified by a PCR using the chromosomal DNA of strain Er as the template, both the forward and the reverse nucleotide sequences of the 790-bp fragment were detected. DNA inversion of the 790-bp fragment also occurred in Escherichia coli DH5alpha when two compatible plasmids carrying either the 790-bp fragment or the invertase gene were cotransformed into the bacterium. Furthermore, hybrid carotovoricin CGE possessing the tail fiber protein of 68 or 76 kDa exhibited a host range specificity corresponding to that of carotovoricin Era or Erb, respectively. Thus, a DNA inversion altered the C-terminal part of the tail fiber protein of carotovoricin Er, altering the host range specificity of the bacteriocin.     

A simple purification method and morphology and component analyses for carotovoricin Er, a phage-tail-like bacteriocin from the plant pathogen Erwinia carotovora Er.

Carotovoricin Er has been isolated as a phage-tail-like bacteriocin from the plant pathogen Erwinia carotovora Er [Kamimiya, S. et al., (1977), Agric. Biol. Chem. 41, 911-912]. However, the fine morphology and structural composition of carotovoricin Er remained to be studied because a large amount of contracted carotovoricin Er were present in the bacteriocin preparations so far obtained. To obtain intact carotovoricin Er and its major parts, we developed simple and efficient purification methods including the use of sucrose density gradient centrifugation in the presence of 10-20% (v/v) ethanol. Electron microscopy for the purified carotovoricin Er showed the presence of a novel antenna-like structure at the proximal end of the phage-tail-like particle, which consisted of a sheath-and-core part, a baseplate, and tail fibers. Contracted sheath and inner core were purified as hollow cylindrical structures with longitudinal lengths of 69 and 174 nm, respectively, and tail fibers were purified as a fibrous structure with length of 63 nm. SDS-polyacrylamide gel electrophoresis showed the presence of single major proteins of 50, 20, and 68 kDa in the isolated sheath, core, and tail fiber, respectively. Three other minor proteins of 46, 44, and 35 kDa were also identified as the structural proteins of carotovoricin Er, which may be the candidate proteins for the antenna-like and the base plate structures. Thus carotovoricin Er consists of at least 6 protein components.     

Nucleotide sequence of pnl gene from Erwinia carotovora Er

The nucleotide sequence of pnl gene encoding pectin lyase (PNL; EC4.2.2.10)from Erwinia carotovora Er was determined. The structural gene of pnl consisted of 942 base pairs. An open reading frame that could encode a 33,700 dalton polypeptide consisting 314 amino acids was assigned. The molecular size of the polypeptide predicted from the amino acid composition was close to the value of PNL determined in E.carotovora Er. The nucleotide sequence of the 5'-flanking region showed the presence of the consensus sequence of ribosome binding site, Pribnow box and the RNA polymerase recognition site in E.carotovora and Escherichia coli. Between the presumed Pribnow box and the ribosome binding site, two pairs of inverted repeats were found. By comparing the predicted amino acid sequences of pnl, several reported bacterial pectate lyases and Aspergillus niger pectin lyase, short regions of homology were found despite the different substrate specificities of these enzymes.     

Molecular cloning and sequencing of the extracellular pectate lyase II gene from Erwinia carotovora Er.

Erwinia carotovora Er produces three extra-cellular pectate lyases (PL I, II, and III). The gene for pectate lyase II (pelII) of E. carotovora Er was cloned and expressed both in Escherichia coli and E. carotovora Er. Localization experiments in E. coli showed that PL II was exclusively in the cytoplasmic space, while PL II was excreted into the culture medium. The complete nucleotides of the pelII gene were sequenced and found to include one open reading frame of 1122 bp coding for a protein of 374 amino acid residues. From comparison of the N-terminal amino acid sequence between the purified PL II and the deduced protein from the nucleotide sequence we reached the conclusion that the mature protein is composed of 352 amino acids with a calculated molecular weight of 38,169 and is preceded by a typical signal sequence of 22 amino acid residues. PL II had 90.1% and 82.9% homologies with PL I and PL III in amino acid sequence, respectively.     

In vivo cloning of Erwinia carotovora genes involved in the catabolism of hexuronates

Using the RP4::mini-Mu pULB113 plasmid, an RP4 derivative carrying a deleted Mu prophage which allows the plasmid to pick up any chromosomal DNA segment to form R' plasmids, we cloned all of the genes of Erwinia carotovora involved in the catabolism of the hexuronates and in the transport of these substrates. With the R' plasmids we isolated, we performed complementation analysis and found that, in the Erwinia carotovora strain we used, the genes involved in the catabolism of the hexuronates are clustered in four regions of the chromosome. This genetic organization is compared with that of Escherichia coli K-12.     

Comparative analysis of P2-type remnant prophage loci in Xenorhabdus bovienii and Xenorhabdus nematophila required for xenorhabdicin production

The xnp1 remnant P2-type prophage of Xenorhabdus nematophila produces xenorhabdicin that is active against closely related species. Xenorhabdicin had not been characterized previously in other Xenorhabdus species. Here, we show xenorhabdicin production in six different strains of Xenorhabdus bovienii. The sequenced genome of X. bovienii SS-2004 was found to possess a highly conserved remnant P2-type cluster (xbp1). Inactivation of the xbpS1 sheath gene resulted in loss of bacteriocin activity, indicating that the xbp1 locus was required for xenorhabdicin production. xbp1 and xnp1 contain a CI-type repressor, a dinI gene involved in stabilization of ssDNA-RecA complexes and are inducible with mitomycin C, suggesting that both loci are regulated by cleavage of the CI repressor. Both xnp1 and xbp1 lack typical P2-type lysis genes but contain a predicted endolysin gene (enp) that may be involved in cell lysis. The main tail fibers of xnp1 and xbp1 are mosaic structures with divergent C-terminal regions suggesting they differ in host specificity. Several genes encoding C-terminal tail fiber fragments are present in the same position in xnp1 and xbp1. Recombination between the main fiber genes and the C-terminal fragments could potentially expand the host range specificity of xenorhabdicin in the respective strains.     

Subclassification and targeted characterization of prophage-encoded two-component cell lysis cassette

Bacteriophage induced lysis of host bacterial cell is mediated by a two component cell lysis cassette comprised of holin and lysozyme. Prophages are integrated forms of bacteriophages in bacterial genomes providing a repertoire for bacterial evolution. Analysis using the prophage database (http://bicmku.in:8082) constructed by us showed 47 prophages were associated with putative two component cell lysis genes. These proteins cluster into four different subgroups. In this process, a putative holin (essd) and endolysin (ybcS), encoded by the defective lambdoid prophage DLP12 was found to be similar to two component cell lysis genes in functional bacteriophages like p21 and P1. The holin essd was found to have a characteristic dual start motif with two transmembrane regions and C-terminal charged residues as in class II holins. Expression of a fusion construct of essd in Escherichia coli showed slow growth. However, under appropriate conditions, this protein could be over expressed and purified for structure function studies.The second component of the cell lysis cassette, ybcS, was found to have an N-terminal SAR (Signal Arrest Release) transmembrane domain. The construct of ybcS has been over expressed in E.coli and the purified protein was functional, exhibiting lytic activity against E.coli and Salmonella typhi cell wall substrate. Such targeted sequence- structure-function characterization of proteins encoded by cryptic prophages will help understand the contribution of prophage proteins to bacterial evolution.     

Structural and functional organization of the colicin operon in the ColD-CA23 plasmid

The organization of the genes involved in colicin D synthesis was studied. These are colicin, immunity and lysis genes. The nucleotide sequence of the immunity gene, its structural and regulatory regions were determined. This gene was shown to be located next to the colicin gene on the same strand and followed by the lysis gene. When colicin synthesis is induced with mitomycin C the immunity gene is transcribed from the general SOS-dependent promotor as a part of the colicin operon. However it has its own SOS-independent promotor in normal growth conditions. A high homology in amino acid sequences of Co1D lysis protein and that of Co1E1, Co1E2, Co1E3, Co1DF13, Co1A was revealed. A detailed scheme of Co1D-CA23 colicin operon structural organization is suggested.     

Cloning and expression of the Erwinia carotovora subsp. carotovora gene encoding the low-molecular-weight bacteriocin carocin S1

The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of Erwinia carotovora. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn5 insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of Erwinia carotovora subsp. carotovora 89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes, caroS1K (ORF2) and caroS1I (ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in E. carotovora subsp. carotovora Ea1068 and replicated in TH22-10 but could not be expressed in Escherichia coli (JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the E. coli cyclic AMP receptor protein binding site (-312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin S3 and pyocin AP41 genes in Pseudomonas aeruginosa. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity.     

The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage

Pseudomonas aeruginosa produces three types of bacteriocins: R-, F- and S-type pyocins. The S-type pyocin is a colicin-like protein, whereas the R-type pyocin resembles a contractile but non-flexible tail structure of bacteriophage, and the F-type a flexible but non-contractile one. As genetically related phages exist for each type, these pyocins have been thought to be variations of defective phage. In the present study, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin, which are located downstream of the R2 gene cluster on the chromosome of P. aeruginosa PAO1, was analysed in order to elucidate the relationship between the pyocins and bacteriophages. The results clearly demonstrated that the R-type pyocin is derived from a common ancestral origin with P2 phage and the F-type from lambda phage. This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages. The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that both pyocins are not simple defective phages, but are phage tails that have been evolutionarily specialized as bacteriocins. A systematic polymerase chain reaction (PCR) analysis of P. aeruginosa strains that produce various subtypes of R and F pyocins revealed that the genes for every subtype are located between trpE and trpG in the same or very similar gene organization as for R2 and F2 pyocins, but with alterations in genes that determine the receptor specificity.     

Chromosomal mapping in Erwinia carotovora subsp. carotovora with the IncP plasmid R68::Mu.

Conjugational gene transfer was established in Erwinia carotovora subsp. carotovora SCRI193 by using plasmid R68::Mu c+ to mobilize the chromosome into multiply mutant recipients. It was observed that although the plasmid alone mobilized markers randomly at a frequency of ca. 10(-5) chromosomal recombinants per donor, the presence of a Mu prophage on the chromosome of the donor increased the frequency of mobilization of markers adjacent to the prophage by up to 10-fold. Using this system it was possible to order 17 chromosomal mutations. The behavior of Mu in E. carotovora subsp. carotovora was also studied.     

Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157:H7 strain derived from the Sakai outbreak

Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains. Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected. We have determined the complete nucleotide sequence of the prophage VT1-Sakai. The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome. The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously. The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21. The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported. These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai. Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages.     

The chromosomal origin of replication (oriC) of Erwinia carotovora.

The chromosomal DNA replication origin (oriC) of the plant pathogen Erwinia carotovora has been isolated and sequenced. The minimal E. carotovora oriC regional functional in Escherichia coli is a 374 base pair region located on a 7.9 kilobase pair SalI fragment which also contains a functional asnA gene. Differences between the nucleotide sequence of the minimal origin regions of E. carotovora and those of E. coli and Salmonella typhimurium are clustered nucleotide substitutions, with regions of complete homology, up to 19 base pairs long, between the three origins. Nine GATC sites are found in the minimal origin, and all are conserved. In contrast, the region toward asnA from the minimal origin shows little clustering and the differences occur mainly every third nucleotide, suggesting that this region is a protein coding region.     

Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes. In the EHEC strains, as well as in other VT-producing E. coli strains, the toxins are encoded by lysogenic bacteriophages. The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments. We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data. To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al. (J. Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical. These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements. An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments.     

The TGV transgenic vectors for single-copy gene expression from the Escherichia coli chromosome

Plasmid-based cloning and expression of genes in Escherichia coli can have several problems: plasmid destabilization; toxicity of gene products; inability to achieve complete repression of gene expression; non-physiological overexpression of the cloned gene; titration of regulatory proteins; and the requirement for antibiotic selection. We describe a simple system for cloning and expression of genes in single copy in the E. coli chromosome, using a non-antibiotic selection for transgene insertion. The transgene is inserted into a vector containing homology to the chromosomal region flanking the attachment site for phage lambda. This vector is then linearized and introduced into a recombination-proficient E. coli strain carrying a temperature-sensitive lambda prophage. Selection for replacement of the prophage with the transgene is performed at high temperature. Once in the chromosome, transgenes can be moved into other lysogenic E. coli strains using standard phage-mediated transduction techniques, selecting against a resident prophage. Additional vector constructs provide an arabinose-inducible promoter (P(BAD)), P(BAD) plus a translation-initiation sequence, and optional chloramphenicol-, tetracycline-, or kanamycin-resistance cassettes. These Transgenic E. coli Vectors (TGV) allow drug-free, single-copy expression of genes from the E. coli chromosome, and are useful for genetic studies of gene function.     

Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex

We have determined the genomic sequences of four virulent myophages, Bcep1, Bcep43, BcepB1A, and Bcep781, whose hosts are soil isolates of the Burkholderia cepacia complex. Despite temporal and spatial separations between initial isolations, three of the phages (Bcep1, Bcep43, and Bcep781, designated the Bcep781 group) exhibit 87% to 99% sequence identity to one another and most coding region differences are due to synonymous nucleotide substitutions, a hallmark of neutral genetic drift. Phage BcepB1A has a very different genome organization but is clearly a mosaic with respect to many of the genes of the Bcep781 group, as is a defective prophage element in Photorhabdus luminescens. Functions were assigned to 27 out of 71 predicted genes of Bcep1 despite extreme sequence divergence. Using a lambda repressor fusion technique, 10 Bcep781-encoded proteins were identified for their ability to support homotypic interactions. While head and tail morphogenesis genes have retained canonical gene order despite extreme sequence divergence, genes involved in DNA metabolism and host lysis are not organized as in other phages. This unusual genome arrangement may contribute to the ability of the Bcep781-like phages to maintain a unified genomic type. However, the Bcep781 group phages can also engage in lateral gene transfer events with otherwise unrelated phages, a process that contributes to the broader-scale genomic mosaicism prevalent among the tailed phages.     

Isolation of a Gallic Acid-producing Microorganism with Sake Cake Medium and Production of Gallic Acid

Carotovoricin Er has been isolated as a phage-tail-like bacteriocin from the plant pathogen Erwinia carotovora Er [Kamimiya, S. et al., (1977), Agric. Biol. Chem. 41, 911-912]. However, the fine morphology and structural composition of carotovoricin Er remained to be studied because a large amount of contracted carotovoricin Er were present in the bacteriocin preparations so far obtained. To obtain intact carotovoricin Er and its major parts, we developed simple and efficient purification methods including the use of sucrose density gradient centrifugation in the presence of 10-20% (v/v) ethanol. Electron microscopy for the purified carotovoricin Er showed the presence of a novel antenna-like structure at the proximal end of the phage-tail-like particle, which consisted of a sheath-and-core part, a baseplate, and tail fibers. Contracted sheath and inner core were purified as hollow cylindrical structures with longitudinal lengths of 69 and 174 nm, respectively, and tail fibers were purified as a fibrous structure with length of 63 nm. SDS-polyacrylamide gel electrophoresis showed the presence of single major proteins of 50, 20, and 68 kDa in the isolated sheath, core, and tail fiber, respectively. Three other minor proteins of 46, 44, and 35 kDa were also identified as the structural proteins of carotovoricin Er, which may be the candidate proteins for the antenna-like and the base plate structures. Thus carotovoricin Er consists of at least 6 protein components.