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An anaerobic continuous-flow hydrogen fermentor was operated at a hydraulic retention time of 8 h using condensed molasses fermentation solubles (CMS) substrate of 40 g-COD/L. Serum bottles were used for seed micro-flora cultivation and batch hydrogen fermentation tests (CMS substrate concentrations of 10–160 g-COD/L). Three hydrogen-producing bacterial strains Clostridium sporosphaeroides F52, Clostridium tyrobutyricum F4 and Clostridium pasteurianum F40 were isolated from the seed fermentor and used as the seeding microbes in single and mixed-culture cultivations for determining their hydrogen productivity. These strains possessed specific hydrogenase genes that could be detected from CMS-fed hydrogen fermentors and were major hydrogen producers. C. pasteurianum F40 was the dominant strain with a high hydrogen production rate while C. sporosphaeroides F52 may play a main role in degrading carbohydrate and glutamate. These strains could be co-cultivated as a symbiotic mixed-culture process to enhance hydrogen productivity. C. pasteurianum F40 or C. tyrobutyricum F4 co-culture with the glutamate-utilizing strain C. sporosphaeroides F52 efficiently enhanced hydrogen production by 12–220% depending on the substrate CMS concentrations.

Mesorhizobium sp. F28 was isolated from a wastewater treatment system in a polyacrylonitrile (PAN) fibre factory for its ability to convert acrylonitrile by a nitrile hydratase (NHase)–amidase (AMase) system. This strain completely degraded acrylonitrile at concentrations as high as 18.4 mM, and exhibited 192 U mg −1 dcw NHase activity for the conversion of acrylonitrile to acrylamide. The cobalt-containing NHase purified from the strain F28 was moderately thermostable, keeping full activity up to 45 °C; it also had optimum activity between pH 7.0 and 7.5. Acrylonitrile, propionitrile, isobutyronitrile, and benzonitrile were good substrates for the NHase of Mesorhizobium sp. F28. The affinity of the NHase for acrylonitrile ( K m = 0.53 mM) was greater than that of bacteria mentioned by other researchers before.

An anaerobic continuous-flow fermentation system with condensed molasses fermentation soluble (CMS) from monosodium glutamate production as substrate was developed for the biohydrogen production. The production rate of the system was increased, while hydraulic retention time (HRT) was varied from 12 to 3 h and the optimal hydrogen production rate was obtained at HRT 3 h. The major bacterial composition of each HRT was analyzed by 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE). Acidaminococcus sp. that could evolve hydrogen from glutamate was the only hydrogen producer and could be also detected from substrate. Hydrogenase mRNA-targeted reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that except HRT 3 h, two clusters of clostridial hydrogenase gene sequences were obtained. RT-PCR-DGGE analysis was employed to illustrate the active clostridial strains confirming that clostridial strains were not present at HRT 3 h. Acidaminococcus sp. existing in substrate might be the major strain that contributes to hydrogen production at HRT 3 h.

By using hydrogenase gene-targeted polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR), the predominant clostridial hydrogenase that may have contributed to biohydrogen production in an anaerobic semi-solid fermentation system has been monitored. The results revealed that a Clostridium pasteurianum-like hydrogenase gene sequence can be detected by both PCR and RT-PCR and suggested that the bacterial strain possessing this specific hydrogenase gene was dominant in hydrogenase activity and population. Whereas another Clostridium saccharobutylicum-like hydrogenase gene can be detected only by RT-PCR and suggest that the bacterial strain possessing this specific hydrogenase gene may be less dominant in population. In this study, hydrogenase gene-targeted fluorescence in situ hybridization (FISH) and flow cytometry analysis confirmed that only 6.6% of the total eubacterial cells in a hydrogen-producing culture were detected to express the C. saccharobutylicum-like hydrogenase, whereas the eubacteria that expressed the C. pasteurianum-like hydrogenase was 25.6%. A clostridial strain M1 possessing the identical nucleotide sequences of the C. saccharobutylicum-like hydrogenase gene was then isolated and identified as Clostridium butyricum based on 16S rRNA sequence. Comparing to the original inoculum with mixed microflora, either using C. butyricum M1 as the only inoculum or co-culturing with a Bacillus thermoamylovorans isolate will guarantee an effective and even better production of hydrogen from brewery yeast waste.

φLf is a filamentous phage (39) specifically infecting Xanthomonas campestris pv. campestris, a gram-negative pathogen causing black rot in cruciferous plants. It is similar to other filamentous phages in morphology, having a single-stranded circular DNA genome (6,008 nucleotides [nt]), producing RF (replicative form) during DNA replication and propagating without lysis of the host (26, 43). However, several interesting properties different from those of other filamentous phages have been found. First, it has a mechanism to integrate its RF DNA into the host chromosome (11, 15). Second, its origin of viral strand replication is contained within the gene coding for the replication initiation protein (pII) instead of the major intergenic region (IR) as is the case in other filamentous phages (16). Third, its pII possesses sequence domains conserved in the superfamily I Rep proteins of the rolling-circle-replicating replicons, a superfamily not including the proteins of other filamentous phages (19). So far, 10 genes have been assigned to the φLf viral strand. These genes, in the order gII-gX-gV-gVII-gIX-gVIII-gIII-gVI-gI-gXI, organized similarly to those of the Escherichia coli filamentous phages Ff, IKe, and 12-2 (26, 30), were predicted to code for proteins pII, pX, pV, pVII, pIX, pVIII, pIII, pVI, pI, and pXI, respectively. pII, pX, and pV are required for DNA replication (19, 44); pVII and pIX are thought to form the protein coat in conjunction with pVIII, pIII, and pVI (20, 21, 44); pI and pXI are presumably involved in phage morphogenesis (6). We have previously isolated filamentous phage φXv from X. campestris pv. vesicatoria (18). Comparative study of φLf and φXv revealed that (i) they are strictly host specific, each phage being able to infect only its own host, not other X. campestris pathovars; (ii) most regions of their DNA exhibit sequence identity, as demonstrated by Southern hybridization; (iii) each of their antisera can cross-react with the other phage particles, indicating that these phages are closely related, and (iv) when RF DNA or single-stranded DNA (ssDNA) from these phages was electroporated into the nonhost Xanthomonas cells, authentic phage particles were produced, indicating that host specificity is eliminated by skipping the early steps of infection (18). Furthermore, we have shown that when φXv is propagated in the presence of cloned φLf gIII, coding for the adsorption protein, the progeny phage particles produced are able to infect both X. campestris pv. campestris and X. campestris pv. vesicatoria (20). In this study, we revised the φLf gIII coding region from the one assigned previously (45), sequenced the gIII gene of φXv, and demonstrated the interchangeability of gIII between φLf and φXv.

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 Pac I, Pme I, and Swa I 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 Pac I, Pme I, and Swa I 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-Ceu I site, one pig cluster required for yellow pigmentation, and nine auxotrophic markers were determined, using mutants isolated by mutagenesis with Tn 5 (pfm)CmKm. This transposon contains a polylinker with sites for several rare-cutting restriction endonucleases located between the chloramphenicol resistance and kanamycin resistance (Km r ) 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-Km r segment from Tn 5 (pfm)CmKm. This is the first map for X. campestris and would be useful for genetic studies of this and related Xanthomonas species.

ORF95 in the filamentous phage φLf 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 gII-gX-gV-gVII-gIX-gVIII-gIII-gVI have been identified for φLf.

AIMS: To isolate phages against extensively drug resistant Acinetobacter baumannii (XDRAB) and characterize the highest lytic capability phage as a model to evaluate the potential on phage therapy. METHODS AND RESULTS: Eight phages were isolated from hospital sewage and showed narrow host spectrum. Phage φkm18p was able to effectively lyse the most XDRAB. It has a dsDNA genome of 45 kb in size and hexagonal head of about 59 nm in diameter and no tail. Bacterial population decreased quickly from 10(8) CFU ml(-1) to 10(3) CFU ml(-1) in 30 min by φkm18p. The 185 kDa lysis protein encoded by φkm18p genome was detected when the extracted protein did not boil before SDS-PAGE; it showed that the lysis protein is a complex rather than a monomer. Phage φkm18p improved human lung epithelial cells survival rates when they were incubated with A. baumannii. Combination of phages (φkm18p, φTZ1 and φ314) as a cocktail could lyse all genotype-varying XDRAB isolates. CONCLUSION: Infections with XDRAB are extremely difficult to treat and development of a phage cocktails therapy could be a therapeutic alternative in the future. Phage φkm18p is a good candidate for inclusion in phage cocktails.

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.

phi Lf is a filamentous bacteriophage of Xanthomonas campestris pv. campestris, which can integrate its genome into the host chromosome. The nucleotide sequence of an EcoRV-SphI fragment (1018 bp) from the phi Lf replicative form DNA was determined. Four contiguous open reading frames (ORFs), orf98-orf43-orf38-orf42, were revealed. ORFs 98 and 42 were identified as the genes encoding a single-stranded DNA-binding protein (Sbp) and a major coat protein, respectively. Sbp was purified and found to bind with a high affinity to ssDNA prepared from phi Lf phage particles. The major coat protein showed sequence features similar to those of the typical major coat proteins of other filamentous phages. However, it appears to be synthesized as a mature product, similar to the situation with Pf3 but different from that with other filamentous phage major coat proteins which are synthesized as pre-coats and are subject to post-translational processing. The M(r)s, estimated by SDS-PAGE, of both the Sbp (10.9K) and the major coat protein (4.1K) coincide with the values deduced from the nucleotide sequences. ORFs 43 and 38 are proposed to be the genes encoding two minor coat proteins. The order of these four genes is similar to that found in the Escherichia coli filamentous phages.

Molecular biological approaches were developed to monitor the potential biohydrogen-producing clostridia in an anaerobic semisolid fermentation system that used brewery yeast waste as the fermentation substrate. The denaturing gradient gel electrophoresis with 16S rDNA gene-targeted polymerase chain reaction (PCR) analysis was employed to confirm the existence of clostridia in the system. Remarkably, reproducible nucleotide sequences of clostridia were obtained from different hydrogen production stages by using hydrogenase gene-targeted reverse transcription (RT)-PCR. These RNA-based information suggested that the predominant hydrogen-producing strains possess either a specific Clostridium pasteurianum-like or a specific Clostridium saccharobutylicum-like hydrogenase sequence. Comparison of the hydrogenase gene-targeted sequence profiles between PCR and RT-PCR revealed that the specific C. pasteurianum-like hydrogenase harboring bacterial strains were dominant in both mRNA and bacterial population level. On the other hand, the specific C. saccharobutylicum-like hydrogenase harboring strains expressed high level of hydrogenase mRNA but may not be dominant in population. Furthermore, quantitative real-time RT-PCR analysis showed the expression pattern of the clostridial hydrogenase mRNA and may serve as an activity index for the system.

One viral strand of φLf, 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 φLf-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 φLf 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.

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.

A 1170-nucleotide fragment of φLf DNA was sequenced. This fragment contains an open reading frame, ORF367, encoding a protein of 367 amino acids (aa) (36710 Da). ORF367 is located downstream from the gene encoding the major coat protein (gVIIIp) and a Rho-independent termination signal. Sequence analysis revealed that the gene product has a Gly-rich domain (70 aa) at the center and a hydrophobic region (26 aa) at the C terminus. These structural features suggest that ORF367 may encode the adsorption protein of φLf.

Background/purpose Interlaboratory comparison of pulsed-field gel electrophoresis (PFGE) patterns is difficult. A key reference of standardized PFGE protocol for Acinetobacter baumannii may address this issue. This study aimed to determine restriction enzymes with rare cutting sites on A baumannii genomes and evaluate their cost-effectiveness, discriminatory power, and interlaboratory consistence of band assignments. Methods There were 42 A baumannii isolates collected, including nine from three hospital outbreaks and 33 sporadic isolates. The numbers of cutting sites for the restriction enzymes were explored using the "Restriction Digest and PFGE" program. The cost-effectiveness for PFGE analysis was evaluated for the tested restriction enzymes, while its discriminatory ability was expressed through a discriminatory index and 95% confidence interval. The interlaboratory consistence of band assignments was evaluated for the 42 A baumannii isolates. Results ApaI was the most cost-effective restriction enzyme for a PFGE protocol for A baumannii . Both AscI and AsiSI were reasonable in terms of costs. ApaI, AscI, and AsiSI exhibited similar discriminatory indices. ApaI generated more than 40 fragments that were close and not easy to resolve, resulting in less consistence of band assignments. AscI and AsiSI generated 10–20 fragments that were clearly resolved, resulting in higher consistence of band assignments. AscI exhibited a close discriminatory power to that of AsiSI and at half of the cost of AsiSI for PFGE analysis. Conclusion We recommend AscI as the primary enzyme and AsiSI as the secondary enzyme for standardizing the PFGE protocol and interlaboratory comparisons of A baumannii .