Functional characterization and transcriptional analysis of degQ of Xanthomonas campestris pathovar campestris.

High-temperature-requirement protein A (HtrA) family proteins play important roles in controlling protein quality and are recognized as virulence factors in numerous animal and human bacterial pathogens. The role of HtrA family proteins in plant pathogens remains largely unexplored. Here, we investigated the HtrA family protein, DegQ, in the crucifer black rot pathogen Xanthomonas campestris pathovar campestris (Xcc). DegQ is essential for bacterial attachment and full virulence of Xcc. Moreover, the degQ mutant strain showed increased sensitivity to heat treatment and sodium dodecyl sulfate. Expressing the intact degQ gene in trans in the degQ mutant could reverse the observed phenotypic changes. In addition, we demonstrated that the DegQ protein exhibited chaperone-like activity. Transcriptional analysis displayed that degQ expression was induced under heat treatment. Our results contribute to understanding the function and expression of DegQ of Xcc for the first time and provide a novel perspective about HtrA family proteins in plant pathogen.

SsrA and SmpB have multifaceted physiological roles in the black rot pathogen Xanthomonas campestris pathovar campestris.

SsrA and SmpB are known to play important roles in translational quality control and are essential for virulence in many human and animal pathogenic bacteria. The physiological roles and contribution of SsrA and SmpB to plant pathogen are unclear. Here, we present evidence to show that ssrA and smpB are involved in pathogenesis of Xanthomonas campestris pathovar campestris, the cause of black rot diseases in crucifers. The ssrA and smpB mutants exhibited defects in bacterial attachment, cell motility, and extracellular enzyme activity. The mutation of ssrA and smpB also resulted in a reduction in temperature tolerance. These altered phenotypes of the ssrA and smpB mutants could be complemented to wild-type levels by the intact ssrA and smpB genes. This is the first demonstration of the roles of SsrA and SmpB in phytopathogen.

Expression and function of clpS and clpA in Xanthomonas campestris pv. campestris.

ATP-dependent proteases (FtsH, Lon, and Clp family proteins) are ubiquitous in bacteria and play essential roles in numerous regulatory cell processes. Xanthomonas campestris pv. campestris is a Gram-negative pathogen that can cause black rot diseases in crucifers. The genome of X. campestris pv. campestris has several clp genes, namely, clpS, clpA, clpX, clpP, clpQ, and clpY. Among these genes, only clpX and clpP is known to be required for pathogenicity. Here, we focused on two uncharacterized clp genes (clpS and clpA) that encode the adaptor (ClpS) and ATPase subunit (ClpA) of the ClpAP protease complex. Transcriptional analysis revealed that the expression of clpS and clpA was growth phase-dependent and affected by the growth temperature. The inactivation of clpA, but not of clpS, resulted in susceptibility to high temperature and attenuated virulence in the host plant. The altered phenotypes of the clpA mutant could be complemented in trans. Site-directed mutagenesis revealed that K223 and K504 were the amino acid residues critical for ClpA function in heat tolerance. The protein expression profile shown by the clpA mutant in response to heat stress was different from that exhibited by the wild type. In summary, we characterized two clp genes (clpS and clpA) by examining their expression profiles and functions in different processes, including stress tolerance and pathogenicity. We demonstrated that clpS and clpA were expressed in a temperature-dependent manner and that clpA was required for the survival at high temperature and full virulence of X. campestris pv. campestris. This work represents the first time that clpS and clpA were characterized in Xanthomonas.

Functional Characterization and Transcriptional Analysis of clpP of Xanthomonas campestris pv. campestris.

The caseinolytic protease (Clp) system is essential for survival under stress conditions and for virulence in several pathogenic bacteria. Xanthomonas campestris pv. campestris (Xcc) is a plant pathogen which causes black rot disease in crucifers. In this study, the Xcc clpP gene which is annotated to encode the proteolytic core of Clp was characterized. Mutation of clpP resulted in susceptibility to high temperature and puromycin stresses. Site-directed mutagenesis revealed that S105, H130, and D179 are critical amino acid residues for ClpP function in puromycin tolerance. Inactivation of clpP also revealed an attenuation of virulence on the host plant and a reduction in the production of extracellular cellulase, mannanase, pectinase, and protease. The affected phenotypes of the clpP mutant could be complemented to wild-type levels by the intact clpP gene. Transcriptional analysis revealed that expression of clpP is induced under heat shock condition.

The clpX gene plays an important role in bacterial attachment, stress tolerance, and virulence in Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris is a bacterial pathogen and the causal agent of black rot in crucifers. In this study, a clpX mutant was obtained by EZ-Tn5 transposon mutagenesis of the X. campestris pv. campestris. The clpX gene was annotated to encode ClpX, the ATP-binding subunit of ATP-dependent Clp protease. The clpX mutant exhibited reduced bacterial attachment, extracellular enzyme production and virulence. Mutation of clpX also resulted in increased sensitivity to a myriad of stresses, including heat, puromycin, and sodium dodecyl sulfate. These altered phenotypes of the clpX mutant could be restored to wild-type levels by in trans expression of the intact clpX gene. Proteomic analysis revealed that the expression of 211 proteins differed not less than twofold between the wild-type and mutant strains. Cluster of orthologous group analysis revealed that these proteins are mainly involved in metabolism, cell wall biogenesis, chaperone, and signal transduction. The reverse transcription quantitative real-time polymerase chain reaction analysis demonstrated that the expression of genes encoding attachment-related proteins, extracellular enzymes, and virulence-associated proteins was reduced after clpX mutation. The results in this study contribute to the functional understanding of the role of clpX in Xanthomonas for the first time, and extend new insights into the function of clpX in bacteria.

Biofilm formation of beta-hemolytic group G Streptococcus dysgalactiae subspecies equisimilis isolates and its association with emm polymorphism.

Biofilm formation has been well known as a determinant of bacterial virulence. Group G Streptococcus dysgalactiae subspecies equisimilis (SDSE), a relevant pathogen with increasing medical importance, was evaluated for the biofilm-forming potential. Microtiter plate assay was used to assess the most feasible medium for group G SDSE to form a biofilm. Among 246 SDSE isolates examined, 46.7%, 43.5%, 33.3%, and 26.4% of isolates showed moderate or strong biofilm-forming abilities using tryptic soy broth (TSB), brain heart infusion broth (BHI), Todd-Hewitt broth (THB), and C medium with 30 mM glucose (CMG), respectively. The addition of glucose significantly increased the biofilm-forming ability of group G SDSE. FCT (fibronectin-collagen-T-antigen) typing of SDSE was first undertaken and 11 FCT types were found. Positive associations of stG10.0 or negative associations of stG245.0, stG840.0, and stG6.1 with biofilm-forming ability of SDSE were, respectively, found. This was the first investigation demonstrating biofilm-forming potential in clinical group G SDSE isolates; also, some significant associations of biofilm-forming ability with certain emm types were presented.

XCC2366, A Gene Encoding A Putative TetR Family Transcriptional Regulator, is Required for Acriflavin Resistance and Virulence of Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot disease in cruciferous plants. The XCC2366 gene product is annotated as a protein belonging to the TetR family of transcriptional regulators. In this study, we evaluated the function and expression of the XCC2366 gene. Mutational analysis demonstrated that XCC2366 is involved in the resistance to acriflavin and is necessary for virulence in Xcc. In addition, the XCC2366 transcription initiation site was mapped at nucleotide A, 63 nucleotide upstream of the XCC2366 translation start codon. Furthermore, transcriptional analysis revealed that the expression of XCC2366 is induced in the presence of acriflavin. Reporter assay also showed that XCC2366 regulates its own expression under acriflavin-supplemented condition. To the best of our knowledge, acriflavin resistance-related gene in the crucifer pathogen Xcc was characterized for the first time.

Functional characterization and transcriptome analysis reveal multiple roles for prc in the pathogenicity of the black rot pathogen Xanthomonas campestris pv. campestris.

Gram-negative phytopathogenic Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in crucifers. The ability of Xcc to incite this disease in plants depends on a number of factors, including exopolysaccharides, extracellular enzymes and biofilm production. In this study, transposon mutagenesis led to identification of the prc gene, encoding a tail-specific protease, which plays a role in Xcc pathogenesis. Mutation of prc resulted in decreased virulence, extracellular protease production and bacterial attachment, with restoration to the levels of wild type by the intact prc gene. From subsequent quantitative RT-PCR analysis and reporter assay, the major extracellular protease gene prt1, biofilm-related gene galE encoding a UDP-galactose 4-epimerase and two putative adhesin genes (yapH and XC_4290 encoding autotransporter-like protein H and hemagglutinin, respectively) were found to be reduced in the prc mutant. Results of transcriptome profiling of Xcc wild type and prc mutant by RNA sequencing (RNA-Seq) showed that mutation of prc in Xcc leads to alteration in the transcriptional levels (more than twofold) of 91 genes. These differentially expressed genes were associated with a wide range of biological functions such as carbohydrate transport and metabolism, cell wall/membrane biogenesis, posttranslational modification, protein turnover and chaperones, inorganic ion transport and metabolism and signal transduction mechanisms. The results of this study facilitate the functional understanding of and provide new information about the regulatory role of prc.

Genomic analysis of Staphylococcus phage Stau2 isolated from medical specimen.

Stau2 is a lytic myophage of Staphylococcus aureus isolated from medical specimen. Exhibiting a broad host range against S. aureus clinical isolates, Stau2 is potentially useful for topical phage therapy or as an additive in food preservation. In this study, Stau2 was firstly revealed to possess a circularly permuted linear genome of 133,798 bp, with low G + C content, containing 146 open reading frames, but encoding no tRNA. The genome is organized into several modules containing genes for packaging, structural proteins, replication/transcription and host-cell-lysis, with the structural proteins and DNA polymerase modules being organized similarly to that in Twort-like phages of Staphylococcus. With the encoded DNA replication genes, Stau2 can possibly use its own system for replication. In addition, analysis in silico found several introns in seven genes, including those involved in DNA metabolism, packaging, and structure, while one of them (helicase gene) is experimentally confirmed to undergo splicing. Furthermore, phylogenetic analysis suggested Stau2 to be most closely related to Staphylococcus phages SA11 and Remus, members of Twort-like phages. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed 14 structural proteins of Stau2 and N-terminal sequencing identified three of them. Importantly, this phage does not encode any proteins which are known or suspected to be involved in toxicity, pathogenicity, or antibiotic resistance. Therefore, further investigations of feasible therapeutic application of Stau2 are needed.

Distribution of virulence factors and association with emm polymorphism or isolation site among beta-hemolytic group G Streptococcus dysgalactiae subspecies equisimilis.

Distribution of virulence factors and association with emm polymorphism or isolation site among beta-hemolytic group G Streptococcus dysgalactiae subspecies equisimilis. Streptococcus dysgalactiae subspecies equisimilis (SDSE), the dominant human pathogenic species among group G streptococci, is the causative agent of several invasive and non-invasive diseases worldwide. However, limited information is available about the distribution of virulence factors among SDSE isolates, or their association with emm types and the isolation sites. In this study, 246 beta-hemolytic group G SDSE isolates collected in central Taiwan between February 2007 and August 2011 were under investigation. Of these, 66 isolates were obtained from normally sterile sites and 180 from non-sterile sites. emm typing revealed 32 types, with the most prevalent one being stG10.0 (39.8%), followed by stG245.0 (15.4%), stG840.0 (12.2%), stG6.1 (7.7%), and stG652.0 (4.1%). The virulence genes lmb (encoding laminin-binding protein), gapC (glyceraldehyde 3-phosphate dehydrogenase), sagA (streptolysin S), and hylB (hyaluronidase) existed in all isolates. Also, 99.2% of the isolates possessed slo (streptolysin O) and scpA (C5a peptidase) genes. In addition, 72.8%, 14.6%, 9.4%, and 2.4% of the isolates possessed the genes ska (streptokinase), cbp (putative collagen-binding protein, SDEG_1781), fbp (putative fibronectin-binding protein, SDEG_0161), and sicG (streptococcal inhibitor of complement), respectively. The only superantigen gene detected was spegg (streptococcus pyrogenic exotoxin G(dys) ), which was possessed by 74.4% of the isolates; these isolates correlated with non-sterile sites. Positive correlations were observed between the following emm types and virulence genes: stG10.0 and stG840.0 with spegg, stG6.1 and stG652.0 with ska, and stG840.0 with cbp. On the other hand, negative correlations were observed between the following: stG245.0, stG6.1, and stG652.0 types with spegg, stG10.0 with ska, and stG10.0, stG245.0, and stG6.1 types with cbp. The prevalence of emm types of SDSE in central Taiwan was investigated for the first time. Moreover, the distribution of virulence factors among beta-hemolytic group G SDSE isolates, as well as their association with emm types or isolation sites were also examined.

Antibiotic susceptibility pattern and erythromycin resistance mechanisms in beta-hemolytic group G Streptococcus dysgalactiae subspecies equisimilis isolates from central Taiwan.

BACKGROUND/PURPOSE: Information concerning antibiotics susceptibilities of beta-hemolytic group G Streptococcus dysgalactiae subspecies equisimilis (SDSE) clinical isolates in central Taiwan was limited. METHODS: Totally, 246 SDSE isolates were collected from mainly five regional hospitals, from February 2007 to August 2011. Disk diffusion method, broth microdilution method, and clindamycin induction test (D test) were respectively performed according to the guidelines of the Clinical and Laboratory Standards Institute. Polymerase chain reaction was used to detect the corresponding erythromycin resistance genes. RESULTS: All isolates were susceptible to penicillin, cefotaxime, and vancomycin. The rate of erythromycin resistance was 24.0% (59/246), whereas that of clindamycin resistance was 12.2% (30/246). The resistance rates of isolates from different hospitals varied from 15.0% to 45.5% for erythromycin and from 7.1% to 36.4% for clindamycin. For erythromycin-resistant SDSE isolates, three different phenotypes with resistance to macrolides (M), lincosamides (L), and type B streptogramins (SB) were observed: M (49.2%), constitutive MLSB (cMLSB, 35.6%), and inducible MLSB (iMLSB, 15.3%). All M phenotypic isolates carried mefA. The most prevalent genotypes among cMLSB and iMLSB phenotypic isolates were ermB, followed by ermTR. One isolate with cMLSB phenotype carried both ermB and ermTR, whereas one isolate with iMLSB phenotype carried both ermB and ermC. CONCLUSION: This is the first trial investigating the antimicrobial susceptibility pattern and erythromycin resistance mechanisms of beta-hemolytic group G SDSE isolates in central Taiwan. The resistance rates for both erythromycin and clindamycin varied significantly among hospitals located in this area and should be monitored continuously in the future.

Identification, epidemiological relatedness, and biofilm formation of clinical Chryseobacterium indologenes isolates from central Taiwan.

BACKGROUND: The clinical impact of Chryseobacterium indologenes infection is increasing; nevertheless, most studies had been conducted in northern Taiwan, but rarely in central Taiwan. METHODS: Using 16S rRNA gene sequencing, 34 isolates of C. indologenes were identified at the Central Region Hospital Alliance between 2007 and 2011. Vitek 2 and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) methods were compared for the feasibility to identify this bacterium. Drug susceptibility test, biofilm formation, and pulsed-field gel electrophoresis (PFGE) were also performed. RESULTS: All isolates were collected from hospitalized patients with an average age of 70.8 ± 18.5 years. The most prevalent sample was urine (50.0%), followed by sputum (32.4%). The accuracy rate of species-level identification reached 94.1% using the Vitek 2 method and 85.3% using the MALDI-TOF MS method. All of the isolates were resistant to gentamicin, amikacin, ceftriaxone, chloramphenicol, colistin, and imipenem, but completely susceptible to minocycline. While analyzing biofilm-forming ability, 38.2% (13/34) of C. indologenes isolates displayed a positive phenotype using the Luria-Bertani (LB) medium. However, 80.0% (4/5) of invasive isolates were biofilm producers. Based on PFGE analysis, several clusters were found, and the possible intrahospital spread of this bacterium in this area could not be excluded. CONCLUSION: Both Vitek 2 and MALDI-TOF MS methods showed good ability in the determination of C. indologenes. Among the examined drugs, minocycline was the most potent one. As many as 38.2% C. indologenes isolates showed biofilm-forming ability. PFGE analyses revealed the possible intrahospital transmission of this bacterium in central Taiwan.

The galU gene of Xanthomonas campestris pv. campestris is involved in bacterial attachment, cell motility, polysaccharide synthesis, virulence, and tolerance to various stresses.

Uridine triphosphate (UTP)-glucose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.9) is an enzyme that catalyzes the formation of uridine diphosphate (UDP)-glucose from UTP and glucose-1-phosphate. GalU is involved in virulence in a number of animal-pathogenic bacteria since its product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharide and exopolysaccharide. However, its function in Xanthomonas campestris pv. campestris, the phytopathogen that causes black rot in cruciferous plants, is unclear. Here, we characterized a galU mutant of X. campestris pv. campestris and showed that the X. campestris pv. campestris galU mutant resulted in a reduction in virulence on the host cabbage. We also demonstrated that galU is involved in bacterial attachment, cell motility, and polysaccharide synthesis. Furthermore, the galU mutant showed increased sensitivity to various stress conditions including copper sulfate, hydrogen peroxide, and sodium dodecyl sulfate. In addition, mutation of galU impairs the expression of the flagellin gene fliC as well as the attachment-related genes xadA, fhaC, and yapH. In conclusion, our results indicate involvement of galU in the virulence factor production and pathogenicity in X. campestris pv. campestris, and a role for galU in stress tolerance of this crucifer pathogen.

Identification, characterization, and biofilm formation of clinical Chryseobacterium gleum isolates.

Chryseobacterium gleum is not commonly isolated from clinical source(s). Using 16S rRNA gene sequencing, we identified 15 C. gleum isolates from the Central Region Hospital Alliance, Taiwan, which were all misidentified: 14 as Chryseobacterium indologenes and 1 as Elizabethkingia meningoseptica using the Vitek 2 GN card. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a rapid and clinically applicable method, was evaluated for the identification of C. gleum, and the rate of species or probable species level identification reached 13.3% and 86.6%, respectively. Using pulsed-field gel electrophoresis analysis, all C. gleum isolates from central Taiwan were found to be epidemiologically unrelated. The most prevalent sample was urine (35.7%, 5/14), followed by sputum (28.6%, 4/14), whereas 1 isolate was from an unknown source. All of the isolates were susceptible to minocycline, 93.3% susceptible to trimethoprim/sulfamethoxazole, but were completely or highly resistant to the other drugs examined. Biofilm-forming ability was observed in 40.0% (6/15) isolates using the Luria-Bertani broth. To the best of our knowledge, this is the first focusing on exploring clinical C. gleum isolates.

Functional characterization and transcriptional analysis of galE gene encoding a UDP-galactose 4-epimerase in Xanthomonas campestris pv. campestris.

The Gram-negative plant pathogen Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers, a disease that causes tremendous agricultural loss. In this study, the Xcc galE gene was characterized. Sequence and mutational analysis demonstrated that the Xcc galE encodes a UDP-galactose 4-epimerase (EC 5.1.3.2), which catalyzes the interconversion of UDP-galactose and UDP-glucose. Alanine substitution of the putative catalytic residues (Ser124, Tyr147, and Lys151) of GalE caused loss of epimerase activity. Further study showed that the Xcc galE mutant had reduced biofilm formation ability. Furthermore, reporter assays revealed that galE transcription exhibits a distinct expression profile under different culture conditions, is subject to catabolite repression, and is positively regulated by Clp and RpfF. In addition, the galE transcription initiation site was mapped. This is the first time that UDP-galactose 4-epimerase has been characterized in the crucifer pathogen Xcc.

Identification and epidemiological relatedness of clinical Elizabethkingia meningoseptica isolates from central Taiwan.

BACKGROUND: Elizabethkingia meningoseptica is an opportunistic pathogen. Identification of E. meningoseptica based on conventional methods is rather labor- and time-consuming. The information on epidemiological relatedness and microbiological characteristics of E. meningoseptica isolates from central Taiwan was limited. METHODS: Forty E. meningoseptica isolates identified by conventional methods were collected by the Central Laboratory of Central Region Hospital Alliance between 2007 and 2011. The amplification of 16S ribosomalDNA gene by polymerase chain reaction with species-specific or universal primers following DNA sequencing was used as a standard identification method. The feasibility of Vitek 2 GN card was also evaluated. Some clinical information of the patients and the drug susceptibilities and epidemiological relatedness of the isolates were analyzed. RESULTS: For the 40 isolates, 39 E. meningoseptica and one Chryseobacterium indologenes were identified using 16S rDNA sequencing. Among the 39 isolates, all could be identified using species-specific primers, whereas only 84.6% could be identified by Vitek 2 GN card with excellent discrimination. All E. meningoseptica isolates were susceptible to minocycline but resistant to many drugs examined including ceftazidime, amikacin, colistin, and imipenem. The pulsed field gel electrophoresis (PFGE) patterns demonstrated that most isolates were quite genetic diversity. The patients had average age of 72.2 ± 14.5 years old (excluded one child patient of 1 year old) and 79.5% of patients were male. Twenty-three patients (59.0%) had underlying diseases. CONCLUSION: The designed species-specific primers could be used to identify E. meningoseptica with 100% of specificity and sensitivity, whereas the Vitek 2 GN card showed considerable ability in E. meningoseptica identification. The PFGE patterns showed that most isolates were genetic diversity enough to exclude the possibility of intrahospital spread.

Association of X-ray repair cross-complementing-6 genotypes with childhood leukemia.

BACKGROUND: The Non-homologous end-joining repair gene XRCC6/Ku70 plays an important role in the repair of DNA double-strand breaks (DSBs), and has been found to be involved in the carcinogenesis of many types of cancers including oral, prostate, breast and bladder cancer. However, the contribution of XRCC6 to childhood leukemia has yet to be studied. In the present study, we investigated the association of XRCC6 genotypes with the risk of childhood leukemia. MATERIALS AND METHODS: Two hundred and sixty-six patients with childhood leukemia and an equal number of age-matched healthy controls recruited in Central Taiwan, were genotyped investigating these polymorphisms' association with childhood leukemia. RESULTS: As for XRCC6 promoter T-991C, patients carrying the TC genotype had a significantly increased risk of childhood leukemia compared with the TT wild-type genotype [odds ratio (OR)=2.30, 95% confidence interval (CI)=1.38-3.84, p=0.0047]. Meanwhile, the genotypes of XRCC6 promoter C-57G, A-31G and intron3 were not statistically associated with childhood leukemia risk. CONCLUSION: Our findings suggest that the XRCC6 genotype could serve as a predictor of childhood leukemia risk and XRCC6 could serve as a target for personalized medicine and therapy.

Identification, clinical aspects, susceptibility pattern, and molecular epidemiology of beta-haemolytic group G Streptococcus anginosus group isolates from central Taiwan.

No literature is available on the prevalence and clinical aspects of beta-haemolytic group G Streptococcus anginosus group in central Taiwan. In this study, we used 16S rRNA gene sequencing and 16S-23S rDNA intergenic spacer sequencing (where necessary) as the gold standard for molecular identification. Twenty-seven S. anginosus group isolates were identified from 273 beta-haemolytic GGS isolates collected from patients in central Taiwan between February 2007 and August 2011. Of the 27 isolates, 22 were S. anginosus and 5 were Streptococcus constellatus. The 3 commercial methods, Rapid ID 32 Strep, API 20 Strep, and Vitek 2 GP card, identified 77.8%, 40.7%, and 37.0% of S. anginosus group isolates, respectively, with acceptable %ID or probability level. All the S. constellatus isolates possessed the lmb gene (encoding laminin-binding protein); however, none of the S. anginosus isolates possessed this gene. All the 27 isolates were susceptible to penicillin. Five S. anginosus group isolates (18.5%) were resistant to erythromycin. The resistance genes, ermB and mefA, were detected in 3 (2 S. anginosus and 1 S. constellatus) and 2 (2 S. anginosus) isolates, respectively. Pulsed field gel electrophoresis showed that most S. anginosus group isolates were genetically diverse. This is the first study to evaluate 3 commercial methods for the identification of beta-haemolytic group G S. anginosus group species, and only the Rapid ID 32 Strep system showed considerable ability. The clinical aspects, susceptibility pattern, and molecular epidemiology of beta-haemolytic group G S. anginosus group isolates from central Taiwan were also first presented.

GsmR, a response regulator with an HD-related output domain in Xanthomonas campestris, is positively controlled by Clp and is involved in the expression of genes responsible for flagellum synthesis.

In prokaryotes, two-component signal transduction systems, consisting of a histidine kinase and a response regulator, play a critical role in regulating a range of cellular functions. A recent study suggests that XCC3315, a response regulator with a CheY-like receiver domain attached to an uncharacterized HD-related output domain (HDOD domain), plays a role in the general stress response of the Gram-negative bacterium Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in cruciferous plants. Here, we demonstrated genetically that XCC3315, designated as gsmR (general stress and motility regulator), is involved in the expression of genes responsible for flagellum synthesis, including rpoN2, flhF, flhB, and fliC. Site-directed mutagenesis revealed that Glu9 and Arg100 in the receiver domain and Gly205, Asp263, His287, Trp298 and His311 in the HDOD are critical amino acids for GsmR function in cell motility regulation. The gsmR transcription initiation site was mapped. Promoter analysis and gel retardation assay revealed that the expression of gsmR is positively controlled by the global transcriptional regulator Clp in a direct manner, and is subject to catabolite repression. Our findings not only extend the previous work on Clp regulation to show that it influences the expression of gsmR in Xcc, but are also the first to characterize the expression of this response regulator gene in this phytopathogen. Furthermore, GsmR is the first HDOD-containing protein of bacteria in which key amino acids have been experimentally identified and characterized.

Wide host range and strong lytic activity of Staphylococcus aureus lytic phage Stau2.

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 × 10(10) 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 × 10(9) out of 2 × 10(12) 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.