Search

Erwinia amylovora strains formed yellow colonies on minimal agar medium MM2 containing asparagine and copper sulfate (MM2Cu), in contrast to a white morphology on minimal agar without copper salt. Additionally, the colonies were mucoid to various extents. The yellow color was characteristic for the fire blight pathogen, including strains from raspberry and from other unusual host plants, and was used to establish a novel plating technique for identification of E. amylovora. The new identification method was especially superior to semi-selective media with sucrose when natural levan-deficient strains were assayed. No growth of E. amylovora was observed for the similar medium MM1 containing 2 mM CuSO4, due to its low content of as paragine. Identification by colony morphology on MM2 agar with copper was confirmed by staining the bacterial capsules with FITC-labeled lectin from Abrus precatorious, a compound which has a high affinity for galactose residues, the main sugar in the capsular exopolysaccharide amylovoran of E. amylovora. Other plant-associated bacteria usually did not produce the typical colony morphology of E. amylovora on MM2 agar with copper. Furthermore, those cells were not stained with the Abrus lectin. Capsule staining was also observed for weakly mucoid strains of E. amylovora, but not for strains with mutations affecting amylovoran synthesis. The secretion of fluorescent compounds by Pseudomonas syringae pathovars and even growth of any other bacterial colonies adjacent to E. amylovora could interfere with the formation of typical yellow colonies on MM2Cu, which could be white in case of dense plating. After screening for white colonies on LB agar, E. amylovora was identified in extracts from Cotoneaster leaves and in bark from apple trees with fire blight symptoms by its yellow growth pattern on MM2Cu agar and by capsule staining. The proposed selective medium gives a clear signal, is easy to prepare, does not contain dyes or any compounds toxic to humans, and can also detect E. amylovora strains deficient in levan synthesis.

Bacteria were isolated from necrotic apple and pear tree tissue and from dead wood in Germany and Austria as well as from pear tree exudate in China. They were selected for growth at 37 °C, screened for levan production and then characterized as Gram-negative, facultatively anaerobic rods. Nucleotide sequences from 16S rRNA genes, the housekeeping genes dnaJ, gyrB, recA and rpoB alignments, BLAST searches and phenotypic data confirmed by MALDI-TOF analysis showed that these bacteria belong to the genus Gibbsiella and resembled strains isolated from diseased oaks in Britain and Spain. Gibbsiella-specific PCR primers were designed from the proline isomerase and the levansucrase genes. Acid secretion was investigated by screening for halo formation on calcium carbonate agar and the compound identified by NMR as acetic acid. Its production by Gibbsiella spp. strains was also determined in culture supernatants by GC/MS analysis after derivatization with pentafluorobenzyl bromide. Some strains were differentiated by the PFGE patterns of SpeI digests and by sequence analyses of the lsc and the ppiD genes, and the Chinese Gibbsiella strain was most divergent. The newly investigated bacteria as well as Gibbsiella querinecans, Gibbsiella dentisursi and Gibbsiella papilionis, isolated in Britain, Spain, Korea and Japan, are taxonomically related Enterobacteriaceae, tolerate and secrete acetic acid. We therefore propose to unify them in the species Gibbsiella acetica sp. nov.

We identified a compound in culture supernatants of Erwinia species, such as Erwinia amylovora, E. pyrifoliae, E. billingiae, E. tasmaniensis, E. persicina and E. rhapontici absorbing at 340 nm, which was associated before with the yellow pigment produced by E. amylovora on media containing copper ions. The compound was purified from E. tasmaniensis strain Et1/99 supernatants by chromatography on Dowex-1 and Dowex-50 columns and identified by HPLC/MS and NMR analysis as 6-thioguanine (6TG). Its signal at 167 Da matched with the expected molecular mass. By random mutagenesis with miniTn5, we obtained mutants defective in the genes for pyrimidine and purine metabolism. A specific gene cluster with ycf genes described by us before, absent in the corresponding region of Escherichia coli, was identified in the genome sequence of three Erwinia species and named tgs region for thioguanine synthesis. Clones of the tgs gene cluster promoted 6TG synthesis and secretion in E. coli, when the bacteria were grown in minimal medium supplemented with amino acids. 6TG was bacteriostatic for E. coli and Salmonella typhimurium strains, with cell growth resumed after prolonged incubation. Similar results were obtained with P. agglomerans strains. Bacteria from the genus Pectobacterium were barely and Rahnella or Gibbsiella species were not inhibited by 6TG. Adenine and guanine relieved the toxic effect of 6TG on E. coli. Non-producing strains were fully virulent on host plants. 6TG synthesis may help erwinias to interfere with growth of some microorganisms in the environment.

Erwinia amylovora produces a compound with an absorbance maximum at 340 nm that forms a yellow colored complex with copper. This compound was identified as 6-thioguanine, a guanine analogue that is used in chemotherapy and the treatment of inflammatory bowel disease. Synthesis of 6-thioguanine could be linked to five genes common in Erwinia genomes, but missing in related genera. Expression of Erwinia tasmaniensis genes tgsA-D in Escherichia coli was sufficient for heterologous production of 6-thioguanine. Transfer of the four biosynthetic E. tasmaniensis genes did not enhance resistance of E. coli against 6-thioguanine. Bacterial and synthetic 6-thioguanine have a strong growth inhibitory effect on many bacteria, such as E. coli or a number of Pantoea agglomerans isolates. While this inhibition of competing species might provide an advantage to Erwinia, further biological functions of 6-thioguanine cannot be excluded. A direct link between 6-thioguanine synthesis of E. amylovora and its pathogenicity was not observed, as two 6-thioguanine negative mutants produced as severe symptoms on apple and pear shoots as did producing strains.

Fire blight caused by the Gram-negative bacterium Erwinia amylovora can be controlled by antagonistic microorganisms. We characterized epiphytic bacteria isolated from healthy apple and pear trees in Australia, named Erwinia tasmaniensis, and the epiphytic bacterium Erwinia billingiae from England for physiological properties, interaction with plants and interference with growth of E. amylovora. They reduced symptom formation by the fire blight pathogen on immature pears and the colonization of apple flowers. In contrast to E. billingiae, E. tasmaniensis strains induced a hypersensitive response in tobacco leaves and synthesized levan in the presence of sucrose. With consensus primers deduced from lsc as well as hrpL, hrcC and hrcR of the hrp region of E. amylovora and of related bacteria, these genes were successfully amplified from E. tasmaniensis DNA and alignment of the encoded proteins to other Erwinia species supported a role for environmental fitness of the epiphytic bacterium. Unlike E. tasmaniensis, the epiphytic bacterium E. billingiae produced an acyl-homoserine lactone for bacterial cell-to-cell communication. Their competition with the growth of E. amylovora may be involved in controlling fire blight.

Bacteria were isolated from flowers and bark of apple and pear trees at three places in Australia. In Victoria, Tasmania and Queensland, strains with white colonies on nutrient agar were screened for dome-shaped colony morphology on agar with sucrose and were found to be closely related by several criteria. The isolates were not pathogenic on apples or pears. They were characterized by a polyphasic approach including microbiological and API assays as well as fatty acid methyl ester analysis, DNA–DNA hybridization and DNA sequencing. For molecular classification, the 16S rRNA cistron and the conserved genes gpd and recA of these bacteria were investigated. Together with other taxonomic criteria, the results of these studies indicate that the bacteria belong to a novel separate species, which we propose to name Erwinia tasmaniensis sp. nov., with the type strain Et1/99T (=DSM 17950T=NCPPB 4357T). From DNA–DNA hybridization kinetics, microbiological characteristics and nucleotide sequence analyses, this species is related to pathogenic Erwinia species, but also to the epiphytic species Erwinia billingiae.

Erwinia amylovora is the causative agent of fire blight, a destructive disease of rosaceous plants. The European population can be divided into several subtypes according to differences in restriction fragment length polymorphism of the XbaI genomic DNA digest analysed with pulsed-field gel electrophoresis. This technique was also used to determine the genetic relatedness of six Croatian isolates to the E. amylovora types found in the countries surrounding Croatia. The isolates belong to the Pt2 pattern type that is characteristic of the East Mediterranean basin. All tested isolates gave essentially the same total cell protein pattern in SDS-polyacrylamide gel electrophoresis. The number of short-sequence DNA repeats in plasmid pEA29 of six isolates was determined by PCR assays and ranged from four to seven. The isolates examined showed high pathogenicity in immature pear fruits. Differences were also revealed in microbiological assays such as amylovoran synthesis, levan formation, siderophore production and colour on coliform medium.

Several epiphytic bacteria isolated from healthy apple and pear flowers were characterized by their phenotypic properties and by DNA sequence comparisons. Selected epiphytes from Australia and South Africa induced hypersensitive response (HR) on tobacco leaves and produced the exopolysaccharide levan. Sequence comparison of 16S rRNA and the housekeeping genes recA and gapDH showed a close relationship of these epiphytes to Erwinia amylovora and to other plant pathogenic bacteria of the genus Erwinia, however, the epiphytes form a separate group and are not pathogenic to apple and pear. Another close relative of E. amylovora, the epiphyte Erwinia billingiae did not produce levan and HR on tobacco, but it synthesized an acyl-homoserine lactone (AHL). The epiphytic strains from Australia and South Africa were antagonists against E. amylovora in assays on immature pear slices and on apple flowers. E. billingiae also showed significantly antagonism against E. amylovora. Using Real-Time PCR and plating samples on selective agar, we demonstrated that the epiphytic isolates provided effective growth suppression of the pathogen when apple flowers were co-inoculated with a high concentration of the epiphytes and low concentrations of E. amylovora. These epiphytes could possibly be used for biological control of fire blight.

Stewart's bacterial wilt and leaf blight of sweet corn and maize is caused by Pantoea stewartii subsp. stewartii. This bacterium can be seed transmitted at a low frequency, so it is subject to quarantine restrictions by many countries. To develop a polymerase chain reaction assay for the identification of this pathogen from field samples and for use in seed health tests, four primer pairs were tested. These were selected from the sequences of hrpS, cpsDE, and the 16S rRNA intergenic transcribed spacer (ITS) region. Under optimal reaction conditions, about 20 and 200 cells of P. stewartii could be detected in pure cultures and leaf lesions, respectively. Other plant-associated enteric bacteria (e.g., P. agglomerans pv. herbicola, P. ananas, Erwinia amylovora, and E. carotovora) either did not produce amplicons or they were not the correct size for P. stewartii. To test further for possible false positives, 29 yellow-pigmented bacteria, mainly other Pantoea spp., were isolated from lesions on old corn leaves and assayed with the ITS primer sets. Except for weak, variable reactions with three P. ananas strains, the bacteria did not test positive. Pulsed field gel electrophoresis (PFGE) was evaluated as an additional test to confirm the identity of P. stewartii. After digestion with SpeI and XbaI, P. stewartii strains could be easily distinguished from related Erwinia and Pantoea spp. and each other.

Transgenic plants, expressing recombinant proteins, are suitable alternatives for the production of relevant immunogens. In the present study, the expression of Puumala virus nucleocapsid protein in tobacco and potato plants (Nicotiana tabacum and Solanum tuberosum) and its immunogenicity was investigated. After infection of leaf discs of SR1 tobacco and tuber discs of potato cv. “Desiree” with the Agrobacterium strain LBA4404 (pAL4404, pBinAR-PUU-S) containing the 1302 bp cDNA sequence of S-RNA segment of a Puumala virus, transgenic tobacco and potato plants expressed the Puumala virus nucleocapsid protein under control of the cauliflower 35S promoter. The recombinant proteins were found to be identical to the authentic Puumala virus nucleocapsid protein as analyzed by immunoblotting. Expression of the nucleocapsid protein was investigated over four plant generations (P to F4) and found to be stable (1 ng/3 μg dried leaf tissue). Transgenic tobacco plants were smaller compared to controls. The transformed potato plants were morphologically similar to control plants and produced tubers as the control potatoes. The S-antigen was expressed at a level of 1 ng protein/5 μg and 1 ng protein/4 μg dried leaf and root tissues, respectively, and remained stable in the first generation of vegetatively propagated potato plants. The immunogenicity of the Puumala virus nucleocapsid protein expressed in Nicotiana tabacum and Solanum tuberosum was investigated in New Zealand white rabbits. They were immunized with leaf extracts from transgenic tobacco and potato plants, and the serum recognized Puumala virus nucleocapsid protein. Transgenic plants expressing hantaviral proteins can thus be used for the development of cost-effective diagnostic systems and for alternative vaccination strategies.

We quantified the expression of the puf and puc operons, which encode pigment binding proteins, in a number of strains of Rhodobacter capsulatus that have defects in genes affecting different steps of bacteriochlorophyll biosynthesis. Our results show that these mutations have a very similar effect on puf and puc expression. This suggests that the reduced expression of genes encoding pigment-binding proteins is due neither to the accumulation of a specific intermediate of bacteriochlorophyll synthesis nor to the lack of an early intermediate, but is rather the result of the lack of a very late intermediate or the bacteriochlorophyll molecule itself.

Several Erwinia amylovora strains isolated in the Netherlands from apple and pear trees and other hosts for fireblight were found to be conditionally deficient in levan synthesis. The levansucrase genes of five strains were cloned via PCR amplification, and the encoded enzyme was expressed in Escherichia coli. Levan was also produced from the cloned genes in high copy number plasmids, when they were transformed into the corresponding levan-deficient strains. The expression was not driven by the promoter in the cloning vector. An lsc transcript was detected by northern blot analysis with an intermediate signal for strain PD207 and, surprisingly, with a strong signal for PD576. The other four strains did not produce a hybridization signal. The nucleotide sequence in front of the promoter region, when assayed for all five deficient strains, was identical with the sequence previously determined from wild type strain Ea7/74. Also strain E8, which is deficient in levan and in amylovoran synthesis, carries an intact lsc -gene. At 28 °C the expression of lsc from the deficient strains was low in several media with various nutrients and sugars. However, when the strains were grown at 18 °C, levan synthesis was partially restored, indicating a temperature-dependent regulation of lsc -expression.

In Rhodobacter capsulatus wild-type strains, the 23S rRNA is cleaved into [16S] and [14S] rRNA molecules. Our data show that a region predicted to form a hairpin-loop structure is removed from the 23S rRNA during this processing step. We have analyzed the processing of rRNA in the wild type and in the mutant strain Fm65, which does not cleave the 23S rRNA. In addition to the lack of 23S rRNA processing, strain Fm65 shows impeded processing of a larger 5.6-kb rRNA precursor and slow maturation of 23S and 16S rRNAs from pre-23S and pre-16S rRNA species. Similar effects have also been described previously for Escherichia coli RNase III mutants. Processing of the 5.6-kb precursor was independent of protein synthesis, while the cleavage of 23S rRNA to generate 16S and 14S rRNA required protein synthesis. We identified a DNA fragment of the wild-type R. capsulatus chromosome that conferred normal processing of 5.6-kb rRNA and 23S rRNA when it was expressed in strain Fm65.

The pigment-binding proteins of Rhodobacter capsulatus are encoded by the polycistronic puf and puc operons. Both operons show higher expression under low oxygen tension than under high oxygen tension in the wild-type strain. The Tn5 mutant strain AH2 shows only low levels of puf and puc mRNA under high and low oxygen tension, indicating that it lacks a gene product required for stimulation of puf and puc gene expression under low oxygen tension. The formation of wild-type levels of photosynthetic complexes and normal oxygen regulation could be restored by the expression in trans of a 1.7 kb fragment of the R. capsulatus wild-type chromosome or by addition of 10 micrograms l-1 vitamin B12 to the growth medium. An open reading frame of 798 nucleotides containing the Tn5 insertion was identified on the 1.7 kb fragment. This open reading frame shows no homology to known genes and has a remarkably high GC content of 76%.

External factors regulate the formation of pigment protein complexes in facultatively photosynthetic bacteria. The puf operon of Rhodobacter capsulatus encodes the pigment binding proteins of the reaction centre and light-harvesting I complex. Here we demonstrate that a single base-pair exchange within a sequence of dyad symmetry upstream of the puf promoter affects both the oxygen regulation and the light regulation of the formation of reaction-centre and light-harvesting I complexes in Rhodobacter capsulatus. Our results imply that effects of oxygen or light ultimately act on the same regulatory DNA sequence, although it is still unknown how these environmental signals are sensed and transmitted to a transcriptional regulator.

Summary In order to determine a possible genomic divergence of Erwinia amylovora‘fruit tree’ and raspberry strains from North America, several isolates were differentiated by pulsed-field gel electrophoresis (PFGE) analysis, the size of short DNA sequence repeats (SSRs) and the nucleotide and deduced amino acid sequences of their hrpN genes. By PFGE analysis European strains are highly related, whereas strains from North America were diverse and were further distinguished by the SSR numbers from plasmid pEA29. The E. amylovora strains from Europe showed identical HrpN sequences in contrast to the American isolates from fruit trees and raspberry. Those were related to each other, but distinguishable by their HrpN patterns. The Asian pear pathogens differed in HrpN among each other and from E. amylovora. Erwinia pyrifoliae isolates and the Erwinia strains from Japan were ordered via their HrpN sequences in agreement with the PFGE patterns. For all three pathogens, dendrograms from PFGE and sequence data indicate an evolutionary diversity within the species in spite of a genetic conservation for parts of the hrpN genes suggesting a long persistence of the Asian pear pathogens in Korea and Japan as well as of fire blight in North America. Some of the divergent American E. amylovora isolates share PFGE patterns with the relatively uniform European strains.

From necrotic tissue of a Nashi pear tree, 24 Erwinia pyrifoliae strains, found to be identical by pulsed-field gel electrophoresis analysis, were isolated. Thirteen strains were not virulent on immature pears and did not induce a hypersensitive response in tobacco leaves. The defective gene hrpL was complemented with intact genes from E. pyrifoliae and Erwinia amylovora .

An array of short-sequence DNA repeats (SSRs) occurs in the plasmid pEA29 of the fire blight pathogen Erwinia amylovora. A large number of "fruit tree" strains, mainly from Central and Western Europe, were screened for their SSR numbers, and the analyses were extended to five raspberry strains from North America and six pear pathogenic Erwinia strains from Japan. The repeat ATTACAGA present in all E. amylovora strains was found to be reiterated 3 to 15 times. The Japanese strains contained the major repeat sequence GGATTCTG, which was reiterated 16 to 24 times. ATTACAGG, which resembles the SSR of E. amylovora, was reiterated two or three times. In a novel approach, sequencing gels were used to visualize the rare occurrence of shorter arrays (down to three repeats) in E. amylovora and the Japanese Erwinia strains. Changes in the repeat numbers in E. amylovora were observed repeatedly when the bacteria had been exposed to stress conditions. The repeat structures of homo- and heteroduplices of PCR-amplified repeats were also analyzed by cleavage of annealed molecules with the single-strand-specific endonuclease from bacteriophage T4. Not only heteroduplexes, but also homoduplexes showed non-matching regions in the SSRs, which could arise from transient formation of loops due to strand slippage during the assays.

In order to find reasons for the absence of fire blight in most countries of the Southern hemisphere, bark samples from apple and pear trees in orchards of the Western Cape region in South Africa were extracted for bacteria which could be antagonistic to Erwinia amylovora. Screening was done in the late growth season and mainly Gram-positive bacteria were isolated. Approximately half of them produced growth inhibition zones on a lawn of E. amylovora. Most isolates were classified as Bacillus megaterium by microbiological assays and in API 50 test systems. They were visualized in the light microscope as non-motile large rods. These strains may not be responsible for the absence of fire blight in orchards, but they may indicate unfavourable climatic conditions for Gram-negative bacteria including E. amylovora. They may reduce the ability of E. amylovora to establish fire blight and could also be useful for application in biological disease control.

Summary Fire blight has been detected recently in several areas of northern Spain and north-eastern Italy. To follow spread of the disease within Europe, more than 120 Erwinia amylovora strains isolated from 1957-2000 in England, France, Germany, The Netherlands, Belgium, Poland, Italy and Spain were assayed using pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA after XbaI digestion. Pattern types Pt1 and Pt4 were found for strains from England. Pt1 was also found in central Europe and eastern France, Pt4 in western France. Pt2 appeared first in Egypt, from where strains with this pattern disseminated north- wards as far as into the Balkans. Pt3 was typical for northern France and Belgium. Strains from Spain dis- played the pattern types Pt3 and Pt4. In Italy, Pt2 was found in the south-eastern areas, Pt3 in the north- eastern areas, and Pt1 was found very recently in orchards adjacent to the Austrian border, together with Pt3. Despite barely controlled trade with fire blight host plants and associated plant products within Europe, the PFGE patterns of the E. amylovora isolates were ordered indicating sequential spread. On the other hand, the appearance of Pt3 in northern Italy and central Spain can be explained by the import of contaminated plants by nurseries.

Several strains of the genus Erwinia, which were isolated in Japan from pear trees with necrotic symptoms that resembled fire blight, and tentatively identified as Erwinia amylovora, were reinvestigated for their relationship to the fire blight pathogen. These isolates produced ooze on slices of immature pears and were mucoid on MM2Cu agar plates, but did not synthesize levan and did not give the expected PCR signals with several primer pairs specific for Erwinia amylovora. The isolates tested positive with PCR primers designed to detect the novel pear pathogen Erwinia pyrifoliae, which was isolated from Nashi pear trees in South Korea. The nucleotide sequence analysis of a DNA fragment preceding the gene cluster for exopolysaccharide synthesis revealed a closer relationship to Erwinia pyrifoliae than to Erwinia amylovora. Plasmid profiles, protein patterns and genomic DNA analysed by PFGE after XbaI and SpeI digestion were different than Erwinia amylovora. Experiments with strains of Erwinia amylovora isolated from raspberry (Rubus sp.), Erwinia mallotivora and Enterobacter pyrinus also did not reveal a relationship between these bacteria and the Japanese Erwinia strains. The latter are not identical to Erwinia pyrifoliae, but possess many similar features to this pathogen that causes Asian pear blight. It is concluded that pathogenic bacteria isolated in Japan from pear trees with symptoms resembling fire blight are possibly different from Erwinia amylovora.

Necrotic symptoms of aerial parts of European and Asian pear trees (Pyrus communis L. and Pyrus pyrifolia Nakai) are often related to a well known bacterial disease of Maloideae: fire blight caused by E. amylovora (8). Nevertheless, Pseudomonas syringae pv. syringae causes pear blast on blossoms. Enterobacter pyrinus has been isolated in Korea (4) from necrotic leaf spots and shoots.

The recently described pathogen Erwinia pyrifoliae, isolated from Nashi pear fruit trees in Korea, resembles the fire blight pathogen Erwinia amylovora in some of its properties. The two pathogens were classified into different species by DNA hybridization kinetics and microbiological criteria. From the nucleotide sequences of the 16S rRNA and the internal transcribed spacer (ITS) region as well as extracellular polysaccharide (EPS)-encoding genes, polymerase chain reaction (PCR) primers were designed that specifically detect E. pyrifoliae but not the fire blight pathogen Erwinia amylovora, and these primers were also applied to identify E. pyrifoliae in necrotic plant material. The genomes of several strains were digested with the restriction enzyme SpeI, and the DNA fragments were analyzed by pulsed-field gel electrophoresis (PFGE). Three groups of patterns could be distinguished for the isolated E. pyrifoliae strains, all different from various E. amylovora strains, which produce a relatively homogeneous PFGE pattern after SpeI digests. Typical fire blight host plants were assayed in a growth chamber or an experimental field for their susceptibility to E. pyrifoliae. A strong preference was found for pear varieties, whereas apple, cotoneaster, hawthorn, or raspberry rarely produced necrotic symptoms. E. pyrifoliae was readily detected in samples from pear orchards in South Korea during 1995 to 1998; however, the Asian pear pathogen was not recovered in necrotic plant tissue from 1999 and 2000.

Most Erwinia amylovora strains form yellow mucoid colonies on solid minimal medium containing asparagine and copper sulfate (MM2Cu). One exception is the strain Ea25/82, which produces white colonies on MM2Cu agar. This strain was transformed with a genomic library of E. amylovora and yellow colonies were recovered. A 1.5-kb fragment was found to complement strain Ea25/82 for color formation, and subsequent sequencing revealed two ORFs. The smaller ORF132(ycfB) overlapped with the end of the larger ORF253(ycfA). The putative protein YcfA shows low homology with K+/Na+ channel transporter ATPases. Resistance genes were inserted in both ORFs, and the E. amylovora strains Ea1/79-YA and Ea1/79-YB were created by site-directed mutagenesis. The mutation in ycfB did not affect color formation, whereas the ycfA mutant formed white colonies on MM2Cu. Sequence analysis of the ycf region in strain Ea25/82 revealed a 1-bp alteration in ycfA and no change in ycfB. Stable complementation of Ea25/82 and Ea1/79-YA, however, required both genes. Carotenoids were not detected in E. amylovora grown in the presence of copper ions. On the other hand, copper-independent secretion of a low-molecular-weight compound with an absorption maximum at 340 nm (CP340) was found for strain Ea1/79, but not for Ea25/82 or the mutant Ea1/79-YA. CP340 formed a complex with copper ions, and complementation with plasmids carrying both ycfA and ycfB restored its release from mutant strains. The compound may be connected with the yellow pigment or function in sensing bacterial population densities.

In Rhodobacter capsulatus the puf operon encodes proteins of the photosynthetic apparatus. The polycistronic puf mRNA is comprised of segments that show differential stability. Here, we show that the rate of decay of the 2.7-kb pufBALMX mRNA species in Escherichia coli depends on the activity of ribonuclease E (RNase E), whereas the degradation of the 0.5-kb pufBA mRNA segment is not affected by a mutation in the rne gene. The RNase E-promoted decay of the pufLMX mRNA depends on the presence of a 1.4-kb pufLM mRNA segment, in which rate-limiting endonucleolytic cleavage was postulated to occur in R. capsulatus. The insertion of 185 bp of this 1.4-kb segment into pufB results in an RNase E-dependent decay of the modified pufBA mRNA segment in E. coli. Our findings suggest that in R. capsulatus an RNase E-like activity is responsible for the rate-limiting endonucleolytic cleavage occurring within the pufLM mRNA segment, whereas the 0.5-kb pufBA mRNA segment is degraded by a different RNase E-independent decay mechanism.

A DNA sequence with dyad symmetry upstream of the transcriptional start of the Rhodobacter capsulatus puf operon, which encodes pigment-binding proteins of the light-harvesting I complex and of the reaction center, has previously been shown to be a protein-binding site (G. Klug, Mol. Gen. Genet. 226:167-176, 1991). When a low-copy-number plasmid with a base pair transition at position -43 within this dyad symmetry in front of the puf structural genes was transferred into a Rhodobacter strain with the puf operon deleted, different phenotypes occurred during cultivation of the transconjugants and the kinetics of the loss of the wild-type phenotype was dependent on the oxygen tension in the culture. After growth for 150 generations, the different phenotypes were stably inherited. The strains having the wild-type phenotype carried the wild-type puf DNA sequence. The original mutation was still present in the strains that showed lighter color. These strains had less light-harvesting II complex in the membrane and showed lower rates of transcription of the puc operon, which encodes the proteins of this complex. This deregulation of puc expression was due to one or more chromosomally located, secondary mutations, not directly to the mutation present on the plasmid. Thus, a single-base-pair transition in the puf upstream region can result in a deregulation of puc expression, suggesting a direct or indirect transcriptional coregulation of both these operons by a common factor.