Transmission and strain variation of Shigella flexneri 4a after mass prophylaxis in a long-stay psychiatric centre.

Shigella flexneri 4a caused sustained outbreaks in a large long-stay psychiatric centre, Taiwan, 2001-2006. Trimethoprim-sulphamethoxazole (SXT) prophylaxis was administered in 2004. We recovered 108 S. flexneri 4a isolates from 83 symptomatic (including one caregiver) and 12 asymptomatic subjects (11 contacts, one caregiver). The isolates were classified into eight antibiogram types and 15 genotypes (six clusters) by using antimicrobial susceptibility testing and pulsed-field gel electrophoresis of NotI-digested DNA, respectively. These characteristics altered significantly after SXT prophylaxis (P < 0·05), with concomitant emergence of SXT-resistant isolates in two antibiogram types. P01 (n = 71), the predominant epidemic genotype, caused infection in two caregivers and five patients under their care; two P01 isolates were recovered from the same patient 6 months apart. These results indicate the importance of sustained person-to-person transmission of S. flexneri 4a by long-term convalescent, asymptomatic or caregiver carriers, and support the emergence of SXT-resistant strains following selective pressure by SXT prophylaxis.

Sequence and copy number of the Xanthomonas campestris pv. campestris gene encoding 16S rRNA.

A 6.7-kb Sau3A1 fragment containing ribosomal RNA genes was cloned from the chromosome of Xanthomonas campestris pv. campestris strain 17 by a PCR-based strategy. Nucleotide sequence was determined for the 16S rRNA gene (1,544 nt). This gene has a G+C content of 54.9% which is similar to the 16S rRNA genes of Escherichia coli and Pseudomonas aeruginosa but different from the value reported for the whole X. campestris chromosome (64%). Sequence alignment revealed that AGGAGG is consensus for ribosome binding, with the internal GGAG to be paired most frequently with the anti-Shine-Dalgarno sequence. This consensus sequence was found in the regions upstream from the initiation codon of 98 Xanthomonas genes among 116 aligned, but not in the remaining genes. This suggests that about 16% of the Xanthomonas genes do not possess typical ribosome binding sites and another mechanism may be required for recognition of correct translation initiation sites. Two rrn operons were detected in Xc17 chromosome by pulsed-field gel electrophoresis and Southern hybridization.

The ori of filamentous phage phi Lf is located within the gene encoding the replication initiation protein.

phi Lf is a filamentous phage of Xanthomonas campestris pv. campestris. In this study, the origin for phi Lf replication was located in a 121-bp TaqI fragment within gII, the gene encoding the replication initiation protein. This fragment, ligating with a GmR catridge, was able to be maintained as a plasmid (pT2) in strain Xc17 with the gII being provided in trans. ssDNA of pT2 was detected in the cells, indicating that pT2 may replicate by a rolling circle replication mechanism. Upon superinfection of Xc17 containing pT2 with phi Lf, transducing particles containing ssDNA of pT2 were released, suggesting the presence of packaging signal in the 121-bp TaqI fragment. This fragment contains a sequence homologous to the nicking sites for superfamily I Rep proteins of the rolling circle-replicating replicons, in concert with the presence of conserved amino acid sequence motifs of the superfamily I Rep proteins in the phi Lf gIIP.

Isolation and characterization of the recA gene of Xanthomonas campestris pv. campestris.

A 1.8-kb NsiI-StuI fragment containing the recA gene of Xanthomonas campestris pv. campestris was cloned by a PCR-based approach and complementation of Escherichia coli HB 101. Sequence analysis of this fragment revealed an ORF (orf343) of 1,032 bp able to encode a protein of 343 amino acids with a calculated MW of 37,021 Da, a size similar to the values detected by in vitro system and Western blotting. It showed 69.6% identity to the E. coli RecA in amino acid sequence. Amino acid residues of the E coli RecA associated with functional activities are conserved in this Xc17 RecA. The recA mutant, L1, constructed by gene replacement, was sensitive to ultraviolet irradiation and methyl methanesulfonate, and deficient in homologous recombination.

A region of the filamentous phage phi Lf genome that can support autonomous replication and miniphage production.

A 2028-bp fragment from the RF DNA of phi Lf, a filamentous phage of Xanthomonas campestris pv. campestris, was maintained autonomously as a minireplicon. Upon superinfection of the cells harboring the minireplicon with phi Lf, transducing miniphage particles were released. The minireplicon contained an open reading frame (ORF346) able to encode a polypeptide of MW39144, which possessed consensus motifs found in the Rep proteins from various sources. These findings suggested ORF346 to be the gene encoding replication initiation protein, gene II (gII) of phi Lf. Upstream to ORF346 were sequences with potential to form hairpin structures and a sequence similar to the integration host factor (IHF) binding site, structures similar to the intergenic region (IR) of the Ff phages. A 15 bp AT-rich core for phi Lf integration was found 37 bp downstream to the IHF binding site.

Effects of large sample loads on column lifetime in preparative-scale liquid chromatography.

In preparative-scale liquid chromatography of proteins, the use of high sample concentration and large sample mass may result in irreversible adsorption to the support surface. This can change the stationary phase characteristics, reduce the capacity, shorten the column lifetime and diminish the economic viability of a particular separation method. Column recycling and regeneration can influence the throughput (mass purified per time unit) and selectivity, and affect the reproducibility. The effects of large sample loads on column lifetime and performance were evaluated for three strong anion-exchange columns: (1) a silica support with a quaternized polyethyleneimine (PEI) coating, (2) a polymeric support with an adsorbed PEI coating which also was quaternized, and (3) a polymeric support with a proprietary quaternary amine stationary phase. The column capacity for proteins was measured by frontal chromatography and monitored as a function of cycle number. The column lifetime was determined by examining chromatographic properties subsequent to the frontal chromatography. The change in protein binding capacity was then compared to the change in nitrate binding capacity. The column performance was evaluated under analytical conditions by measuring the change in resolution of standard protein mixtures.

The gene encoding UDP-glucose pyrophosphorylase is required for the synthesis of xanthan gum in Xanthomonas campestris.

Xanthomonas campestris pv. campestris produces a large quantity of exopolysaccharide, xanthan gum, rendering the colonies mucoid. G76E was a non-mucoid mutant isolated from Xc17 by Tn5 mutagenesis. A 3.0-kb KpnI-EcoRI fragment from the Xc17 chromosome was able to restore mucoid phenotype to G76E. Sequence analysis of the region responsible for the restoration revealed an open reading frame, ORF324, able to encode a polypeptide of 35,232 Da which shows striking similarity to the UDP-glucose pyrophosphorylases from bacteria. The activity of UDP-glucose pyrophosphorylase reduced drastically in G76E was found to be regained in the presence of the cloned 3.0-kb KpnI-EcoRI fragment. In vitro expression of the gene in the S30 transcription/translation system produced a protein of ca. 35 kDa.

Development of children's knowledge about unconsciousness.

Children of ages 5, 6, 7, 8, and 10 years, and adults were asked whether people who are sound asleep and not dreaming could or would: (1) see, hear, listen, notice, think, wish, pretend, and feel things (primary-consciousness activities), (2), know that they are asleep, and know whether they are or are not engaged in primary-consciousness activities such as perceiving and thinking (reflective-consciousness activities), (3) deliberately decide to or try to perform mental or physical actions (control activities). Results indicated that the recognition that people do not engage in conscious mental activities when unconscious is still developing during the late middle-childhood years. We speculate that a developing understanding of consciousness may assist the development of an understanding of unconsciousness and vice versa, and that sensitivity to the phenomenology as contrasted with the content of mental states may be a late acquisition in the theory-of-mind area.

Plasmids carrying cloned fragments of RF DNA from the filamentous phage (phi)Lf can be integrated into the host chromosome via site-specific integration and homologous recombination.

Different regions of RF DNA from the filamentous bacteriophage phiLf were cloned in Escherichia coli vectors that can not be maintained in Xanthomonas. After introduction into X. campestris pv. campestris 17 (Xc17), most of these constructs were found to integrate into the host chromosome, either by recA-dependent homologous recombination or recA-independent site-specific integration. Mutations in himA, which codes for the alpha-subunit of the Integration Host Factor, does not affect the integration. Integration occurs into a chromosomal region which harbors a copy of a defective phage (4445 bp) that shares a high degree of identity with the phiLf genome. While various parts of the 4445-bp region are susceptible to homologous recombination, site-specific integration requires the attB sequence on the chromosome and the phage attP. The attB shows a high level of sequence identity (22 out of 28 bp) to the dif site required for E. coli Xer site-specific recombination, including the 6-bp central region, and 8/11 identity in both the left XerC-binding arm and the right XerD-binding arm, with the innermost 5 nt of the arms forming a dyad symmetry that is also present in dif. The attP has the same central region and shows 10/11 identity to the dif site in the left arm, but the sequence of the right arm is less conserved than that of attB. The smallest regions still capable of mediating integration are a cloned 72-bp phiLf attP-containing sequence and a 51-bp Xc17 attB-containing sequence, which was reinserted into the Xc17 chromosome after the 4445-bp region had been deleted, indicating that accessory sequences are not necessary and that the integrase required for site-specific integration is neither specified by the 4445-bp Xc17 chromosomal region nor encoded by the phiLf genome.

Nucleotide sequence and expression of UDP-glucose dehydrogenase gene required for the synthesis of xanthan gum in Xanthomonas campestris.

Xanthomonas campestris pv. campestris, producing large amounts of exopolysaccharide xanthan gum, has a mucoid phenotype. Strain SD7 was a non-mucoid mutant deficient in UDP-glucose dehydrogenase. A DNA fragment able to complement the mutation of SD7 was cloned from the parental wild-type strain Xc11. Sequence analysis of the region required for the complementation revealed an open reading frame which could encode a polypeptide of 445 amino acids with a calculated molecular weight of 48,432, a size similar to that of the product produced by maxicell. The amino acid sequence had significant homology to that of the GDP-mannose dehydrogenase from Pseudomonas aeruginosa.

Identification of gene VI of filamentous phage phi Lf coding for a 10-kDa minor coat protein.

ORF95 in the filamentous phage phi Lf genome, locating behind gIII, was identified to be the gene (gVI) coding for minor coat protein pVI (95 amino acids, 10,245 dal). It was shown to be virion associated by Western blot analysis of chloroform-treated phage particles. Computer analysis predicted two transmembrane regions for this protein. Since no signal peptide was suggested and the size estimated by SDS-polyacrylamide gel electrophoresis matches that deduced from nucleotide sequence, it appears to be incorporated into the phage particle as its primary translational product. After completion of this study, eight genes organizing into an order of gVII-gX-gV-gVII-gIX-gIII-gIII-gVI have been identified for phi Lf.

Purification and expression of the gene III protein from filamentous phage phi Lf.

The gene III protein (pIII) from phi Lf, a filamentous phage of Xanthomonas campestris pv.campestris, was purified by gel filtration with FPLC. The gIII coding region was amplified by PCR, which was then cloned into pUC18 and expressed in Escherichia coli. The size of both pIII, purified from phage particle and expressed in E. coli, is similar to the value deduced from the nucleotide sequence as shown by Western blot analysis. This is different from the case in Ff phages (f1, fd, and M13), in which the size of pIII observed in SDS-polyacrylamide gel electrophoresis is substantially larger than the deduced value. Upon infection of X. c. pv. vesicatoria carrying cloned phi Lf gIII with phi Xv, a filamentous phage of pv. vesicatoria, the progeny particles in supernatant were able to infect both pv. campestris carrying cloned phi Lf gIII and pv. vesicatoria, indicating that a mixture of authentic phi Xv and chimeric phage consisting of phi Xv DNA and phi Lf pIII was produced. These results suggest pIII to be the adsorption protein required for host recognition.

Sequence analysis and expression of the filamentous phage phi Lf gene I encoding a 48-kDa protein associated with host cell membrane.

One viral strand of phi Lf, a filamentous phage of Xanthomonas campestris pv.campestris, the open reading frame (ORF440) behind gene VI was identified as gene I. This gene codes for pI protein (440 aa, 48 kDa) which was shown to be membrane-bound in the phi Lf-infected host cell by Western blot analysis using the antibody raised against the protein expressed in Escherichia coli. Its predicted amino acid sequence has a nucleotide-binding motif in the N-terminal 97 aa and a membrane-spanning domain (aa 221 to 236). These structural features are characteristic of pIs of several filamentous phages which are transmembrane proteins required for phage assembly. Thus far, nine phi Lf genes have been identified which are organized in the order GII-gX-gV-gVII-gIX-gVIII-gIII-gVI-gI, similar to the genome organization of E. coli filamentous phages.

Characterization of two novel filamentous phages of Xanthomonas.

Two filamentous phages of Xanthomonas campestris pv. vesicatoria and Xanthomonas oryzae pv. oryzae were isolated and designated phi Xv and phi Xo, respectively. They were similar to other filamentous phages of Xanthomonas in (i) shape, (ii) restrictive host specificity, (iii) high stability, (iv) an ssDNA genome, (v) a dsDNA as the replicative form (RF), (vi) propagation without lysis of host cells and (vii) ability to integrate into the host chromosome. These phages showed sequence homology to filamentous phage phi Lf of X. c. pv. campestris. phi Xv was inactivated by antisera against phi Xv, phi Xo and phi Lf, whereas phi Xo and phi Lf were inactivated only by their respective antisera and the anti-phi Xv serum. Both the single-stranded phage DNAs and the RF DNAs of phi Xv, phi Xo and phi Lf were able to transfect X. c. pv. vesicatoria, X. o. pv. oryzae and X. c. pv. campestris. Physical maps of phi Xv and phi Xo were constructed for the RF DNAs. Genome sizes were estimated, based on mapping data, to be 6.8 kb for phi Xv and 7.6 kb for phi Xo, larger than that of the phi Lf genome (6.0 kb). The difference in genome sizes appeared to result from insertions of large DNA fragments. These fragments and the regions mediating integration were localized in the physical maps.

The adsorption protein genes of Xanthomonas campestris filamentous phages determining host specificity.

Gene III (gIII) of phiLf, a filamentous phage specifically infecting Xanthomonas campestris pv. campestris, was previously shown to encode a virion-associated protein (pIII) required for phage adsorption. In this study, the transcription start site for the gene and the N-terminal sequence of the protein were determined, resulting in the revision of the translation initiation site from the one previously predicted for this gene. For comparative study, the gIII of phiXv, a filamentous phage specifically infecting X. campestris pv. vesicatoria, was cloned and sequenced. The deduced amino acid sequences of these two pIIIs exhibit a high degree of identity in their C-terminal halves and possess the structural features typical of the adsorption proteins of filamentous phages: a signal sequence in the N terminus, a long glycine-rich region near the center, and a hydrophobic membrane anchorage domain in the C terminus. The regions between gIII and the upstream gVIII, 128 nucleotides in both phages, are larger than those of other filamentous phages. A hybrid phage of phiXv, consisting of the phiLf pIII and all the other components derived from phiXv, was able to infect X. campestris pv. campestris but not X. campestris pv. vesicatoria, indicating that gIII is the gene specifying host specificity and demonstrating the interchangeability of the pIIIs.

The Xanthomonas campestris gumD gene required for synthesis of xanthan gum is involved in normal pigmentation and virulence in causing black rot.

A cloned 4.1-kb EcoRI fragment from Xanthomonas campestris pv. campestris was previously shown to complement the non-mucoid mutant P22 and increase xanthan gum production after being transformed into the wild-type strain Xc17. The gene responsible for these effects was identified, sequenced, and shown to be the gumD gene which has previously been proposed to encode glucose transferase activity, an enzyme required for adding the first glucose residue to the isoprenoid glycosyl carrier lipid during xanthan synthesis. A gumD mutant, isolated from Xc17 by gene replacement, was shown to possess altered pigment xanthomonadin profiles and exhibit reduced virulence in causing black rot in broccoli. This study appears to be the first to demonstrate that interruption of a gene required for xanthan synthesis can lead to reduced virulence of X. campestris.

Chromosome map of Xanthomonas campestris pv. campestris 17 with locations of genes involved in xanthan gum synthesis and yellow pigmentation.

No plasmid was detected in Xanthomonas campestris pv. campestris 17, a strain of the causative agent of black rot in cruciferous plants isolated in Taiwan. Its chromosome was cut by PacI, PmeI, and SwaI into five, two, and six fragments, respectively, and a size of 4.8 Mb was estimated by summing the fragment lengths in these digests. Based on the data obtained from partial digestion and Southern hybridization using probes common to pairs of the overlapping fragments or prepared from linking fragments, a circular physical map bearing the PacI, PmeI, and SwaI sites was constructed for the X. campestris pv. campestris 17 chromosome. Locations of eight eps loci involved in exopolysaccharide (xanthan gum) synthesis, two rrn operons each possessing an unique I-CeuI site, one pig cluster required for yellow pigmentation, and nine auxotrophic markers were determined, using mutants isolated by mutagenesis with Tn5(pfm)CmKm. This transposon contains a polylinker with sites for several rare-cutting restriction endonucleases located between the chloramphenicol resistance and kanamycin resistance (Kmr) genes, which upon insertion introduced additional sites into the chromosome. The recA and tdh genes, with known sequences, were mapped by tagging with the polylinker-Kmr segment from Tn5(pfm)CmKm. This is the first map for X. campestris and would be useful for genetic studies of this and related Xanthomonas species.