Yi-Hsiung Tseng

Isolation and Characterization of Novel Giant Stenotrophomonas maltophilia Phage φSMA5

ABSTRACT Stenotrophomonas maltophilia is one of the most prevalent opportunistic bacteria causing nosocomial infections. It has become problematic because most of the isolates are resistant to multiple antibiotics, and therefore, development of phage therapy has attracted strong attention. In this study, eight S. maltophilia phages were isolated from clinical samples including patient specimens, catheter-related devices, and wastewater. These phages can be divided into four distinct groups based on host range and digestibility of the phage DNAs with different restriction endonucleases. One of them, designated φSMA5, was further characterized. Electron microscopy showed it resembled Myoviridae, with an isometric head (90 nm in diameter), a tail (90 nm long), a baseplate (25 nm wide), and short tail fibers. The φSMA5 double-stranded DNA, refractory to digestion by most restriction enzymes, was tested and estimated to be 250 kb by pulsed-field gel electrophoresis. This genome size is second to that of the largest phage, φKZ of Pseudomonas aeruginosa. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 25 virion proteins were visualized. N-terminal sequencing of four of them suggested that each of them might have had its N terminus cleaved off. Among the 87 S. maltophilia strains collected in this study, only 61 were susceptible to φSMA5, indicating that more phages are needed toward a phage therapy strategy. Since literature search yielded no information about S. maltophilia phages, φSMA5 appears to be the first reported.

Wide Host Range and Strong Lytic Activity of Staphylococcus aureus Lytic Phage Stau2

ABSTRACT In searching for an alternative antibacterial agent against multidrug-resistant Staphylococcus aureus, we have isolated and characterized a lytic staphylophage, Stau2. It possesses a double-stranded DNA genome estimated to be about 134.5 kb and a morphology resembling that of members of the family Myoviridae. With an estimated latency period of 25 min and a burst size of 100 PFU/infected cell, propagation of Stau2 in liquid culture gave a lysate of ca. 6 × 1010 PFU/ml. It was stable at pH 5 to 13 in normal saline at room temperature for at least 4 weeks and at −85°C for more than 2 years, while 1 × 109 out of 2 × 1012 PFU/ml retained infectivity after 36 months at 4°C. Stau2 could lyse 80% of the S. aureus isolates (164/205) obtained from hospitals in Taiwan, with complete lysis of most of the isolates tested within 3 h; however, it was an S. aureus-specific phage because no lytic infection could be found in the coagulase-negative staphylococci tested. Its host range among S. aureus isolates was wider than that of polyvalent phage K (47%), which can also lyse many other staphylococcal species. Experiments with mice demonstrated that Stau2 could provide 100% protection from lethal infection when a multiplicity of infection of 10 was administered immediately after a challenge with S. aureus S23. Considering these results, Stau2 could be considered at least as a candidate for topical phage therapy or an additive in the food industry.

Clp upregulates transcription of engA gene encoding a virulence factor in Xanthomonas campestris by direct binding to the upstream tandem Clp sites

In Xanthomonas campestris, the causative agent of black rot in crucifers, the endoglucanase level is greatly decreased in the mutant deficient in Clp, a homologue of cyclic AMP receptor protein (CRP). It is established that Clp has the same DNA binding specificity as CRP at positions 5, 6, and 7 (GTG motif) of the DNA half site. In this study, the engA transcription initiation site was determined by the 5′ RACE method, and two consensus Clp‐binding sites, site I and site II centered at −69.5 and −42.5, respectively, were located. Transcriptional fusion assays indicated that Clp greatly activates engA transcription. Site‐directed mutagenesis indicated that position 5 of GTG motif in site II is essential for both DNA‐protein complex formation in electrophoretic mobility shift assays and engA transcription in vivo. In addition, mutation at position 5 of site I drastically reduces the promoter activity, indicating that binding of Clp to site I exerts a synergistic effect on the transcription activation by site II. engA appears to be the first X. campestris gene known to be activated by Clp via a direct binding to the promoter.

Flagellin gene fliC of Xanthomonas campestris is upregulated by transcription factor Clp.

Clp, a homologue of cyclic AMP receptor protein (CRP), of Xanthomonas campestris regulates the expression of many genes. In this study, proteomic analysis showed the amounts of several extracellular proteins in a clp mutant to be reduced, including the flagellin encoded by fliC. Transcriptional fusion assay showed that activity of fliC promoter is reduced by 2.3-fold in clp mutant compared to the wild-type, coincident with the protein levels. The clp mutant is slightly reduced in motility; however, electron microscopy showed no significant change in the monotrichous flagellation. A fleQ homologue with conserved Clp-binding site in the upstream region is present in the fully sequenced X. campestris genome, suggesting that regulation of the flagellar genes is similar to that of Pseudomonas aeruginosa in involving Vfr, the CRP homologue, and FleQ in a cascade manner except that Vfr downregulates fleQ. Concomitant loss of flagellum and motility in fliC mutant and absence of a second homologue in the genome sequence suggest that X. campestris possesses a single flagellin gene, fliC. In addition, mutation of this gene does not affect virulence.

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.

oriC Region and Replication Termination Site, dif, of the Xanthomonas campestris pv. campestris 17 Chromosome

ABSTRACT A 13-kb DNA fragment containing oriC and the flanking genes thdF, orf900, yidC, rnpA, rpmH, oriC, dnaA, dnaN, recF, and gyrB was cloned from the gram-negative plant pathogen Xanthomonas campestris pv. campestris 17. These genes are conserved in order with other eubacterial oriC genes and code for proteins that share high degrees of identity with their homologues, except for orf900, which has a homologue only in Xylella fastidiosa. The dnaA/dnaN intergenic region (273 bp) identified to be the minimal oriC region responsible for autonomous replication has 10 pure AT clusters of four to seven bases and only three consensus DnaA boxes. These findings are in disagreement with the notion that typical oriCs contain four or more DnaA boxes located upstream of the dnaA gene. The X. campestris pv. campestris 17 attB site required for site-specific integration of cloned fragments from filamentous phage φLf replicative form DNA was identified to be a dif site on the basis of similarities in nucleotide sequence and function with the Escherichia coli dif site required for chromosome dimer resolution and whose deletion causes filamentation of the cells. The oriC and dif sites were located at 12:00 and 6:00, respectively, on the circular X. campestris pv. campestris 17 chromosome map, similar to the locations found for E. coli sites. Computer searches revealed the presence of both the dif site and XerC/XerD recombinase homologues in 16 of the 42 fully sequenced eubacterial genomes, but eight of the dif sites are located far away from the 6:00 point instead of being placed opposite the cognate oriC. The differences in the relative position suggest that mechanisms different from that of E. coli may participate in the control of chromosome replication.

Lytic Myophage Abp53 Encodes Several Proteins Similar to Those Encoded by Host Acinetobacter baumannii and Phage phiKO2

ABSTRACT Acinetobacter baumannii is an important Gram-negative opportunistic pathogen causing nosocomial infections. The emergence of multiple-drug-resistant A. baumannii isolates has increased in recent years. Directed toward phage therapy, a lytic phage of A. baumannii, designated Abp53, was isolated from a sputum sample in this study. Abp53 has an isometric head and a contractile tail with tail fibers (belonging to Myoviridae), a latent period of about 10 min, and a burst size of approximately 150 PFU per infected cell. Abp53 could completely lyse 27% of the A. baumannii isolates tested, which were all multiple drug resistant, but not other bacteria. Mg2+ enhanced the adsorption and productivity of, and host lysis by, Abp53. Twenty Abp53 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 47-kDa protein being the predicted major capsid protein. Abp53 has a double-stranded DNA genome of 95 kb. Sequence analyses of a 10-kb region revealed 8 open reading frames. Five of the encoded proteins, including 3 tail components and 2 hypothetical proteins, were similar to proteins encoded by A. baumannii strain ACICU. ORF1176 (one of the tail components, 1,176 amino acids [aa]), which is also similar to tail protein gp21 of Klebsiella phage phiKO2, contained repeated domains similar to those within the ACICU_02717 protein of A. baumannii ACICU and gp21. These findings suggest a common ancestry and horizontal gene transfer during evolution. As phages can expand the host range by domain duplication in tail fiber proteins, repeated domains in ORF1176 might have a similar significance in Abp53.

Mutation of the gene encoding a major outer-membrane protein in Xanthomonas campestris pv. campestris causes pleiotropic effects, including loss of pathogenicity

Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot in crucifers. The xanthan polysaccharide and extracellular enzymes produced by this organism are virulence factors, the expression of which is upregulated by Clp (CRP-like protein) and DSF (diffusible signal factor), which is synthesized by RpfF. It is also known that biofilm formation/dispersal, regulated by the effect of controlled synthesis of DSF on cell-cell signalling, is required for virulence. Furthermore, a deficiency in DSF causes cell aggregation with concomitant production of a gum-like substance that can be dispersed by addition of DSF or digested by exogenous endo-beta-1,4-mannanase expressed by Xcc. In this study, Western blotting of proteins from a mopB mutant (XcMopB) showed Xcc MopB to be the major outer-membrane protein (OMP); Xcc MopB shared over 97 % identity with homologues from other members of Xanthomonas. Similarly to the rpfF mutant, XcMopB formed aggregates with simultaneous production of a gummy substance, but these aggregates could not be dispersed by DSF or endo-beta-1,4-mannanase, indicating that different mechanisms were involved in aggregation. In addition, XcMopB showed surface deformation, altered OMP composition, impaired xanthan production, increased sensitivity to stressful conditions including SDS, elevated temperature and changes in pH, reduced adhesion and motility and defects in pathogenesis. The finding that the major OMP is required for pathogenicity is unprecedented in phytopathogenic bacteria.

Genomic Characterization of the Intron-Containing T7-Like Phage phiL7 of Xanthomonas campestris

ABSTRACT The lytic phage phiL7, which morphologically belongs to the Siphoviridae family, infects Xanthomonas campestris pv. campestris. Nucleotide sequence analysis has revealed that phiL7 contains a linear double-stranded DNA genome (44,080 bp, 56% G+C) with a 3′-protruding cos site (5′-TTACCGGAC-3′) and 59 possible genes. Among the deduced proteins, 32 have homologs with known functions and 18 show no database similarities; moreover, the genes encoding these 18 proteins mostly have varying G+C contents and form clusters dispersed along the genome. Only 39 genes have sequences related (27% to 78%) to those of sequenced genes of X. oryzae pv. oryzae phages, although the genome size and architecture of these Xanthomonas phages are similar. These findings suggest that phiL7 acquired genes by horizontal transfer, followed by evolution via various types of mutations. Major differences were found between phiL7 and the X. oryzae pv. oryzae phages: (i) phiL7 has a group I intron inserted in the DNA polymerase gene, the first such intron observed in Xanthomonas phages; (ii) although infection of phiL7 exerted inhibition to the host RNA polymerase, similar to the situations in X. oryzae pv. oryzae phages Xp10 and Xop411, sequence analysis did not identify a homologue of the Xp10 p7 that controls the shift from host RNA polymerase (RNAP) to viral RNAP during transcription; and (iii) phiL7 lacks the tail fiber protein gene that exhibits domain duplications thought to be important for host range determination in OP1, and sequence analysis suggested that p20 (tail protein III) instead has the potential to play this role.

Constitutive Expression of a Chromosomal Class A (BJM Group 2) β-Lactamase in Xanthomonas campestris

ABSTRACT Sequencing of the upstream region of the β-lactamase gene from Xanthomonas campestris pv. campestris 11 (blaXCC-1) revealed the cognate ampR1 gene (289 amino acids, 31 kDa). It runs divergently from blaXCC-1 with a 100-bp intergenic region (IG) containing partially overlapped promoters with structural features typical of the bla-ampR IG. The deduced AmpR1 protein shows significant identity in amino acid sequence and conserved motifs with AmpR proteins of other species, e.g., of Pseudomonas aeruginosa (58.2% amino acid identity). Results of insertional mutation, complementation tests, and β-lactamase assays suggested that expression of blaXCC-1 was constitutive and dependent on AmpR1. Four bla genes and two ampR genes are present in the fully sequenced X. campestris pv. campestris ATCC 33913 genome, with XCC3039 and XCC3040 considered the analogues of blaXCC-1 and ampR1, respectively. An ampR1 homologue was detected by Southern hybridization in the ampicillin-resistant Xanthomonas strains, which appear to express β-lactamase constitutively. Although the significance remains to be studied, constitutive expression of β-lactamase by a widespread bacterial genus raises environmental concerns regarding the dissemination of resistance genes.