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Aspergillus fumigatus strain 76T-3 formed clear zones on agar plates containing emulsified polyhydroxybutyrate (PHB), polyethylene succinate (PES), polybutylene succinate (PBS), polycaprolactone (PCL), or polylactide (PLA). The strain grew well at 40 °C in Sabouraud Dextrose Broth. Solution-casted PHB films were almost completely degraded after incubation with 76T-3 at 45 °C for 17 h. An extracellular polyester-degrading enzyme was purified from the supernatant of 76T-3 cultures in basal medium containing PHB as the sole carbon source. Zymography results portrayed that the purified enzyme degraded PHB, PES, and PBS but not PCL or PLA. The amino acid sequence obtained from LC-MS/MS identified this enzyme to be a PHB depolymerase with a molecular mass of 57 kDa. The optimal reaction condition for the enzyme was pH 6.4 at 55 °C. The recombinant PHB depolymerase (rPhaZ) expressed in E. coli showed the enzyme can act on PHB only and not on PES or PBS.

The phaC, phaP, phaR, and phaZ genes are involved in the synthesis, accumulation, and degradation of poly-beta-hydroxybutyrate (PHB). These genes encode the PHB synthase, phasin, regulatory protein, and PHB depolymerase, respectively, and are located in the same locus in the genome of Rhodobacter sphaeroides FJ1, a purple nonsulfur bacterium capable of producing PHB. We have previously found that the PhaR protein binds to the promoter regions of phaP, phaR, and phaZ and represses their expression. In this study, we determined that PhaR binds to an 11-bp palindromic sequence, 5'-CTGCN(3)GCAG-3', located at nucleotides -69 to -59 and -97 to -87 relative to the translation start site of phaP. Substitution of the three spacer nucleotides with any three or four nucleotides in this sequence had no effect on PhaR binding, but all other base deletions or substitutions in this sequence abolished its ability to bind PhaR both in vitro and in vivo. These results suggest that PhaR regulates the expression of phaP in R. sphaeroides FJ1.

Thermophilic actinomycetes strains were isolated from various environment in Taiwan and screened for degradation of poly(ethylene succinate) (PES), poly(epsilon-caprolactone) (PCL) and/or poly(beta-hydroxybutyrate) (PHB) by the clear-zone method. Out of 341 strains of thermophilic actinomycetes, 105 isolates were PHB-degraders (30.8%), 198 isolates were PCL-decomposers (58.1%), and 99 isolates could degrade PES (29.0%). Furthermore, 77 isolates could degrade both PHB and PCL (22.6%), 35 isolates could degrade both PHB and PES (10.3%), 81 isolates could degrade both PES and PCL (23.8%) and 31 isolates could degrade the three polyesters used in this study (9.1%). Base on the morphological and chemical characteristics, these 31 isolates belonging to Actinomadura (12.9%), Microbispora (25.8%), Streptomyces (48.4%), Thermoactinomyces (9.7%) and Saccharomonospora genus (3.22%).

Two uvrA-like genes, designated uvrA1 and uvrA2, that may be involved in nucleotide excision repair in Xanthomonas axonopodis pv. citri (X. a. pv. citri) strain XW47 were characterized. The uvrA1 gene was found to be 2,964 bp in length capable of encoding a protein of 987 amino acids. The uvrA2 gene was determined to be 2,529 bp with a coding potential of 842 amino acids. These two proteins share 71 and 39% identity, respectively, in amino acid sequence with the UvrA protein of Escherichia coli. Analyses of the deduced amino acid sequence revealed that UvrA1 and UvrA2 have structures characteristic of UvrA proteins, including the Walker A and Walker B motifs, zinc finger DNA binding domains, and helix-turn-helix motif with a polyglycine hinge region. The uvrA1 or uvrA2 mutant, constructed by gene replacement, was more sensitive to DNA-damaging agents methylmethane sulfonate (MMS), mitomycin C (MMC), or ultraviolet (UV) than the wild type. The uvrA1 mutant was four orders of magnitude more sensitive to UV irradiation and two orders of magnitude more sensitive to MMS than the uvrA2 mutant. The uvrA1uvrA2 double mutant was one order of magnitude more sensitive to MMS, MMC, or UV than the uvrA1 single mutant. These results suggest that UvrA1 plays a more important role than UvrA2 in DNA repair in X. a. pv. citri. Both uvrA1 and uvrA2 genes were found to be constitutively expressed in the wild type and lexA1 or lexA2 mutant of X. a. pv. citri, and treatment of these cells with sublethal dose of MMC did not alter the expression of these two genes. Results of electrophoresis mobility shift assays revealed that LexA1 or LexA2 does not bind to either the uvrA1 or the uvrA2 promoter. These results suggest that uvrA expression in X. a. pv. citri is not regulated by the SOS response system.

We previously identified and characterized a lexA gene from Xanthomonas axonopodis pv. citri. For this study, we cloned and expressed a lexA homologue from X. axonopodis pv. citri. This gene was designated lexA2 , and the previously identified lexA gene was renamed lexA1 . The coding region of lexA2 is 606 bp long and shares 59% nucleotide sequence identity with lexA1 . Analyses of the deduced amino acid sequence revealed that LexA2 has structures that are characteristic of LexA proteins, including a helix-turn-helix DNA binding domain and conserved amino acid residues required for the autocleavage of LexA. The lexA2 mutant, which was constructed by gene replacement, was 4 orders of magnitude more resistant to the DNA-damaging agent mitomycin C at 0.1 μg/ml and 1 order of magnitude more resistant to another DNA-damaging agent, methylmethane sulfonate at 30 μg/ml, than the wild type. A lexA1 lexA2 double mutant had the same degree of susceptibility to mitomycin C as the lexA1 or lexA2 single mutant but was 1 order of magnitude more resistant to methylmethane sulfonate at 30 μg/ml than the lexA1 or lexA2 single mutant. These results suggest that LexA1 and LexA2 play different roles in regulating the production of methyltransferases that are required for repairing DNA damage caused by methylmethane sulfonate. A mitomycin C treatment also caused LexA2 to undergo autocleavage, as seen with LexA1. The results of electrophoresis mobility shift assays revealed that LexA2 does not bind the lexA1 promoter. It binds to both the lexA2 and recA promoters. However, neither LexA2 nor LexA1 appears to regulate recA expression, as lexA1 , lexA2 , and lexA1 lexA2 mutants did not become constitutive for recA transcription and RecA production. These results suggest that recA expression in X. axonopodis pv. citri is regulated by mechanisms that have yet to be identified.

The pilA gene, which encodes the major structure of pili, is required for infection of Xanthomonas axonopodis pv. citri ( X. a. pv. citri) by the filamentous bacteriophage Cf. Two open reading frames (ORFs) located downstream of pilA were cloned and characterized. One 1392-bp ORF encodes a protein of 464 amino acids which shares substantial similarity with pilR of other bacterial species; the second ORF ( orf618), of 1854-bp, shares sequence similarity with pilS. The existence of the pilR-like and pilS-like genes in various X. campestris pathovars indicated that these two genes are well conserved in Xanthomonas. pilR and pilS mutants were constructed by gene replacement. We found that a pilR mutant, resistant to the infection of phage Cf, was unable to synthesize PilA protein; however, the abundance of the PilA protein and of the pilA transcript was markedly increased by the introduction of a plasmid containing the cloned pilR gene. The restoration of the normal pilus-specific sensitivity of this transformed clone to Cf indicated that the pilR gene functions as a transcriptional regulator of pilA. The pilS mutant, however, was susceptible to Cf infection, and the level of pilA expression in this mutant was similar to that of wild-type cells. Promoter analysis of luciferase reporter gene constructs containing the 5' untranslated regions of pilR or pilS genes revealed that, although the pilR and pilS are contiguous in X. a. pv. citri, the two genes are expressed independently, and the strong pilR promoter leads to the accumulation of PilR in X. a. pv. citri, which positively regulates the biosynthesis of PilA. These results revealed the enhanced sensitivity of X. a. pv. citri to phage Cf in the presence of PilR and indicated that the filamentous phage Cf utilize bacterial pili as a receptor site for its infection.

Levels of l exA transcripts are markedly increased upon exposure of Xanthomonas axonopodis pathovar citri ( X. a. pv. citri) to the DNA-damaging agent mitomycin C. Preliminary electrophoretic mobility-shift data led us to propose that binding of LexA protein to the sequence upstream of the lexA coding region is responsible for low promoter activity in the uniduced state. We determined that the LexA protein binds to the region located between the transcription start site and the translation initiation codon of the lexA gene of X. a. pv. citri. Using a DNase I footprinting technique, we identified a 19-bp palindromic sequence, TTAGTAGTAATACTACTAA (TTAGN(11)CTAA), located in this region as the binding sequence for the LexA protein of X. a. pv. citri, and showed that the two halves of the palindrome have to be in the inverted repeat orientation to permit binding of LexA. We also showed that almost any mutation in this sequence, including changes in the length of the spacer region of the palindrome, destroyed its ability to bind LexA both in vitro and in vivo.

A gene cluster containing lexA, recA and recX genes was previously identified and characterized in Xanthomonas campestris pathovar citri (X. c. pv. citri). We have now cloned and sequenced the corresponding regions in the Xanthomonas campestris pv. campestris (X. c. pv. campestris) and Xanthomonas oryzae pathovar oryzae (X. o. pv. oryzae) chromosome. Sequence analysis of these gene clusters showed significant homology to the previously reported lexA, recA and recX genes. The genetic linkage and the deduced amino acid sequences of these genes displayed very high identity in different pathovars of X. campestris as well as in X. oryzae. Immunoblot analysis revealed that the over-expressed LexA protein of X. c. pv. citri functioned as a repressor of recA expression in X. c. pv. campestris, indicating that the recombinant X. c. pv. citri LexA protein was functional in a different X. campestris pathovar. The abundance of RecA protein was markedly increased upon exposure of X. c. pv. campestris to mitomycin C, and an upstream region of this gene was shown to confer sensitivity to positive regulation by mitomycin C on a luciferase reporter gene construct. A symmetrical sequence of TTAGTAGTAATACTACTAA present within all three Xanthomonas lexA promoters and a highly conserved sequence of TTAGCCCCATACCGAA present in the three regulatory regions of recA indicate that the SOS box of Xanthomonas strains might differ from that of Escherichia coli.

Two genes important in DNA repair, recA and lexA, were recently identified in Xanthomonas campestris pathovar citri (X.c. pv. citri). An open reading frame located immediately downstream of lexA and recA has now been isolated from this pathovar and characterized. This 486-bp open reading frame encodes a protein of 162 amino acids and shares substantial sequence similarity with recX of other bacterial species. The X.c. pv. citri RecX protein was overexpressed in Escherichia coli and purified; SDS-polyacrylamide gel electrophoresis revealed a molecular size of 18 kDa for the purified protein. Whereas Northern blot analysis failed to detect recX mRNA in X.c. pv. citri, recX transcripts were detected in this pathovar by reverse transcription and polymerase chain reaction analysis. The increased abundance of recX transcript in X.c. pv. citri revealed that the recX promoter was activated by exposure of cells to DNA-damaging agents. Southern blot and polymerase chain reaction analyses revealed the presence of a recX-related gene in all nine additional X. campestris pathovars tested. The genetic arrangement of lexA-recA-recX was apparent in X. campestris and each of the three genes transcribed from their own promoters.

Filamentous bacteriophages have very strict host specificities. Experiments were performed to investigate whether the A protein of the filamentous phage Cf, which infects Xanthomonas campestris pv. citri but not X. campestris pv. oryzae, is involved in determining Cf's host specificity. The gene encoding the A protein of Cf was cloned and expressed in X. campestris pv. citri. The genomic DNA of another filamentous bacteriophage, Xf, which infects X. campestris pv. oryzae but not X. campestris pv. citri, was then introduced by electroporation into X. campestris pv. citri that had expressed the A protein of Cf. The progeny phages thus produced were able to infect both X. campestris pv. oryzae and X. campestris pv. citri, indicating that the A protein of Cf was incorporated into the viral particles of Xf and conferred upon Xf the ability to infect the host of Cf. Inactivation of the A protein gene abolished the infectivity of Cf. The results of this study indicate that the A protein of Cf is responsible for controlling the host specificity of Cf.

A thermophilic Streptomyces thermoviolaceus subsp. thermoviolaceus isolate 76T-2 that can degrade poly(ε-caprolactone) (PCL) was isolated from soil in Taiwan. Isolate 76T-2 grew well in urea fructose oatmeal medium and exhibited clear zones on agar plates containing PCL, indicating the presence of extracellular PCL depolymerases. The PCL powder present in culture medium was completely degraded within 6 h of culture at 45°C. Two PCL-degrading enzymes were purified to homogeneity from the culture supernatant. The molecular weights of these two enzymes were estimated to be 25 kDa and 55 kDa, respectively. A portion of the N-terminal region of the 25-kDa protein was determined, and the sequence Ala-Asn-Phe-Val-Val-Ser-Glu-Ala thus obtained was identical to that of A64-A71 of the Chi25 chitinase of Streptomyces thermoviolaceus OPC-520. The 25-kDa protein was shown to also degrade chitin, suggesting that isolate 76T-2 has the ability to degrade both PCL and chitin.

The phaC, phaP, phaR, and phaZ genes are involved in the synthesis, accumulation, and degradation of poly-beta-hydroxybutyrate (PHB). These genes encode the PHB synthase, phasin, regulatory protein, and PHB depolymerase, respectively, and are located in the same locus in the genome of Rhodobacter sphaeroides FJ1, a purple nonsulfur bacterium capable of producing PHB. We have previously found that the PhaR protein binds to the promoter regions of phaP, phaR, and phaZ and represses their expression. In this study, we determined that PhaR binds to an 11-bp palindromic sequence, 5'-CTGCN(3)GCAG-3', located at nucleotides -69 to -59 and -97 to -87 relative to the translation start site of phaP. Substitution of the three spacer nucleotides with any three or four nucleotides in this sequence had no effect on PhaR binding, but all other base deletions or substitutions in this sequence abolished its ability to bind PhaR both in vitro and in vivo. These results suggest that PhaR regulates the expression of phaP in R. sphaeroides FJ1.

A cluster of genes encoding polyhydroxybutyrate (PHB) depolymerase (phaZ), PHB synthase (phaC), phasin (phaP), and the regulator protein (phaR) was previously identified in Rhodobacter sphaeroides FJ1 (R. sphaeroides FJ1). In this study, we investigated the role of the PhaR protein on the expression of the pha genes. Immunoblot analysis revealed that the expressions of phaP, phaZ and phaR genes in wild-type cells of R. sphaeroides FJ1 are repressed during the active growth phase, with the exception of phaC. A phaR deletion mutant of R. sphaeroides FJ1 was constructed, and the basal level of phaP and phaZ expression in this mutant was markedly increased. Electrophoretic mobility shift assays demonstrated that PhaR binds to the promoter region of phaP as well as those of phaR and phaZ. These results suggest that the PhaR protein is a repressor of phaP, phaR, and phaZ genes in R. sphaeroides FJ1.

Xanthomonas axonopodis pv. citri (X. axonopodis pv. citri) possesses two lexA genes, designated lexA1 and lexA2. Electrophoretic mobility shift data show that LexA1 binds to both lexA1 and lexA2 promoters, but LexA2 does not bind to the lexA1 promoter, suggesting that LexA1 and LexA2 play different roles in regulating the expression of SOS genes. In this study, we have determined that LexA2 binds to a 14-bp dyad-spacer-dyad palindromic sequence, 5'-TGTACAAATGTACA-3', located at nucleotides -41 to -28 relative to the translation start site of lexA2 of X. axonopodis pv. citri. The two spacer nucleotides in this sequence can be changed from AA to TT without affecting LexA2 binding; all other base deletions or substitutions abolish LexA2 binding. The LexA1 binding sequence in the promoter region of lexA2 is TTAGTACTAAAGTTATAA and is located at -133 to -116, and that in the lexA1 gene is AGTAGTAATACTACT located at nucleotides -19 to -5 relative to the translation start site of lexA1. Any base change in the latter sequence abolishes LexA1 binding.

The lexA gene of Xanthomonas campestris pathovar citri (X.c. pv. citri) was cloned and sequenced. The 639-bp open reading frame encodes a protein of 213 amino acids that shares substantial sequence homology with the products of previously characterized lexA genes, sharing 46% identity with the LexA protein of Escherichia coli. Amino acids required for autocatalytic cleavage of LexA are conserved in the X.c. pv. citri protein, whereas domains thought to mediate DNA binding differ markedly from those of LexA proteins from E. coli and other bacteria. The X.c. pv. citri LexA protein was overexpressed in E. coli, and SDS-polyacrylamide gel electrophoresis revealed a molecular size of 23 kDa for the purified protein. A lexA mutant of X.c. pv. citri was constructed by gene replacement, and the basal level of recA expression in this mutant was shown to be similar to that for wild-type cells exposed to a DNA-damaging agent. These results indicate that LexA functions as a repressor of recA expression in X.c. pv. citri.

Host factors that are important for infection of Xanthomonas campestris pv. citri by the filamentous bacteriophage cf were investigated by transposon mutagenesis with Tn5tac1. A mutant, XT501, that was resistant to cf infection was recovered, showing that the gene inactivated by the transposon is required for infection by the phage but not for cf replication or assembly. A 1.7-kb SacI-ApaI DNA fragment from XT501 containing the bacterial DNA flanking one end of the transposon was cloned and shown to be required for cf infection. Nucleotide sequence analysis of the 1.7-kb fragment reveals the presence of an ORF that encodes a protein of 146 amino acids. This protein shows 42% identity to the type 4 prepilin encoded by the pilA genes of other bacteria. The pilA gene of X. campestris pv. citri is thus essential for infection by the bacteriophage cf.

The abundance of the RecA protein and of recA transcripts was markedly increased on exposure of Xanthomonas campestris pathovar citri to various DNA-damaging agents, including mitomycin C. The promoter sequence responsible for mediating the sensitivity of recA expression to DNA damage was investigated by subcloning a 426-bp restriction fragment of the 5′ untranslated and coding region of the gene into a promoterless vector containing the luxAB genes of Vibrio fischeri. Xanthomonas campestris pv. citri cells transformed with this vector responded to DNA-damaging agents with a marked increase in luciferase activity. Deletion of nucleotides from the 5′ end of the recA fragment inserted into the reporter plasmid revealed that the 58 bp upstream of the transcription initiation site are sufficient to mediate induction of recA expression by mitomycin C.

Summary The antigen-antibody neutralization of filamentous phage Xf showed first-order kinetics within the first 10 minutes. Failure to reverse the neutralization reaction by dilution indicated that a stable combination might be formed between phage and antibody. Antigenicity of Xf was analysed by agar double diffusion and three distinct precipitin lines were observed. For identifying these three antigenic components, Xf particles were dissolved and separated into A-protein and B-protein. The results of agar double diffusion showed that one precipitin line corresponds to the A-protein and the other two precipitin lines are determined by the B-protein.

Summary Twenty-three restriction endonucleases were used to cleave the genome of bacteriophage Cf. BamHI, EcoRI, HaeII, KpnI, PstI, SalI, SmaI and SstII each cut Cf DNA at one site, HindIII, PvuI, PvuII and XhoI cleaved at two sites, BglII cleaved at three sites, HincII cleaved at four sites, HinfI, BstNI, AluI, HpaII and HaeIII cleaved at 14, 19, 20, 24 and 26 sites, respectively, whereas HpaI and XbaI have no cleavage site on the Cf genome. HhaI and TaqI cleaved at more than 20 and more than 8 sites respectively. Based on the sizes of the fragments produced by digestion with these enzymes, Cf DNA was estimated to have 7.62 ± 0.1 kilobase pairs. Using the EcoRI cleavage site as a reference point, nine restriction enzymes, BamHI, BglII, EcoRI, HindIII, KpnI, PstI, PvuII, SstII and XhoI were selected and a detailed physical map of the Cf genome was constructed.