John C. Willison

Genetic Mapping of the Rhodopseudomonas capsulata Chromosome Shows Non-clustering of Genes Involved in Nitrogen Fixation

Summary: The mutant R plasmid pTH10 was used to construct a circular linkage map of the Rhodopseudomonas capsulata B10 chromosome. Mutations affecting nitrogen fixation (nif mutations) were dispersed in several groups on the chromosome. Biochemical analysis of nif mutants allowed identification of the structural gene for the nitrogenase component II or Fe protein (nifH) and a putative regulatory gene, possibly nifA. These two genes appeared closely linked in conjugation experiments, but represented two distinct linkage groups in crosses mediated by gene transfer agent. Other mutants were affected in the synthesis and/or stability of the nitrogenase component I or MoFe protein; synthesis of component II was also affected, but to a lesser extent. In two of these mutants, nitrogenase activity and the content of component I was increased five- to sixfold by the addition of 1 mM-molybdate to the growth medium.

Isolation of an IHF-deficient mutant of a Pseudomonas aeruginosa mucoid isolate and evaluation of the role of IHF in algD gene expression

The role of integration host factor (IHF) in the regulation of alginate synthesis was investigated in a mucoid strain of Pseudomonas aeruginosa (strain CHA) isolated from a cystic fibrosis patient. Escherichia coli strain BL21(DE3) was made IHF-deficient by inactivation of its chromosomal IHF genes, himA and himD, then used as host strain to overproduce P. aeruginosa IHF. The purified recombinant IHF protein was used to determine the affinity of IHF for the two IHF binding sites in the algD promoter. The Kd values were determined to be 130 nM for algD IHF site 2 and about 2 microM for algD IHF site 1. Two IHF-deficient mutants of P. aeruginosa strain CHA were constructed by insertional inactivation of the himA gene, and the activity of the algD promoter was determined using transcriptional fusion with xylE as reporter gene. The expression of algD, the structural gene for GDP-mannose dehydrogenase, was decreased three- to fourfold in the himA mutants under conditions of high salinity and nitrogen limitation. Assays of alginate production by cultures grown on agar plates indicated that the IHF-deficient mutants synthesized 50% less polymer than the mucoid parental strain. These results demonstrate clearly that although IHF is dispensable for alginate production, himA expression is required for full activation of algD expression.

Effectiveness of ultrasound for the destruction of Mycobacterium sp. strain (6PY1).

Ultrasound is widely used to disinfect drinking water and wastewater due to its strong physical and chemical effects on microorganisms. The aim of this study was to investigate the effect of ultrasound on the destruction of Mycobacterium strain 6PY1. Ultrasound waves (20 kHz or 612 kHz) were used to treat aqueous suspensions of Mycobacterium at different volumes, initial bacterial concentrations, and power densities. At the same power density and the same exposure time, sonication at high frequency resulted in a lower destruction of Mycobacterium sp. 6PY1 (35.5%) than sonication at low frequency (93%). The percentage of removal was not significantly affected by the volume of the irradiated suspension (150-300 ml) or the initial cell concentration (2.15 x 10(-3)-1.4 x 10(-2)mg protein L(-1)). At low frequency, the removal percentage of Mycobacterium sp. 6PY1 increased with increasing the power density, with a constant level reached after a certain power density. At high frequency, the removal percentage of Mycobacterium sp. 6PY1 increased with increasing the power density. The mechanism of cell killing was investigated by examining the effects of OH() radical scavengers such as sodium carbonate. At high frequency the presence of sodium carbonate suppressed the removal process. However, at low frequency the removal process was not affected, thus indicating that OH() radicals have a negligible role in this case. The latter result was supported by ten times H(2)O(2) production at high frequency greater than that at low frequency.

Ammonia-dependent growth (Adg-) mutants of Rhodobacter capsulatus and Rhodobacter sphaeroides : comparison of mutant phenotypes and cloning of the wild-type (adgA) genes

Summary: Mutants of Rhodobacter capsulatus deficient in growth on nitrogen sources other than NH4 + were compared with mutants of a similar phenotype isolated from Rhodobacter sphaeroides. In addition to N2 and some amino acids (glutamate, alanine, proline, arginine), mutants of R. sphaeroides and R. capsulatus strain AD2 were unable to utilize NO3 − as sole nitrogen source for growth. Under conditions of nitrogen starvation, mutants of both species lacked the methylammonium (ammonium) uptake system, which was found in the wild-type strains under these conditions. The wild-type (adgA) genes complementing these mutants were isolated from gene banks of the respective species and localized to a 2·9 kb BamHI-SalI fragment in R. sphaeroides and to a 1·7 kb SmaI fragment in R. capsulatus. These two fragments hybridized strongly with each other, showing that they contain homologous sequences. Furthermore, the adgA gene from R. capsulatus fully restored the wild-type phenotype to Adg− mutants of R. sphaeroides and vice versa. Inactivation of the chromosomal adgA gene by insertion of an antibiotic-resistance cassette resulted in a severe inhibition of growth in rich medium and in minimal medium containing NH4 +. This suggests that the adg A gene is required for normal growth under all growth conditions.

Zinc biosorption by the purple non-sulfur bacterium Rhodobacter capsulatus

This paper presents the first report providing information on the zinc (Zn) biosorption potentialities of the purple non-sulfur bacterium Rhodobacter capsulatus. The effects of various biological, physical, and chemical parameters on Zn biosorption were studied in both the wild-type strain B10 and a strain, RC220, lacking the endogenous plasmid. At an initial Zn concentration of 10 mg·L(-1), the Zn biosorption capacity at pH 7 for bacterial biomass grown in synthetic medium containing lactate as carbon source was 17 and 16 mg Zn·(g dry mass)(-1) for strains B10 and RC220, respectively. Equilibrium was achieved in a contact time of 30-120 min, depending on the initial Zn concentration. Zn sorption by live biomass was modelled, at equilibrium, according to the Redlich-Peterson and Langmuir isotherms, in the range of 1-600 mg Zn·L(-1). The wild-type strain showed a maximal Zn uptake capacity (Qm) of 164 ± 8 mg·(g dry mass)(-1) and an equilibrium constant (Kads) of 0.017 ± 0.00085 L·(mg Zn)(-1), compared with values of 73.9 mg·(g dry mass)(-1) and 0.361 L·mg(-1) for the strain lacking the endogenous plasmid. The Qm value observed for R. capsulatus B10 is one of the highest reported in the literature, suggesting that this strain may be useful for Zn bioremediation. The lower Qm value and higher equilibrium constant observed for strain RC220 suggest that the endogenous plasmid confers an enhanced biosorption capacity in this bacterium, although no genetic determinants for Zn resistance appear to be located on the plasmid, and possible explanations for this are discussed.

Role and Evolution of Endogenous Plasmids in Photosynthetic Bacteria

Abstract Endogenous plasmids are found in most strains of Rhodobacter sphaeroides , Rhodobacter capsulatus and Rhodospirillum rubrum , but appear to be less common in other species of purple non-sulphur bacteria. They have also been found in some species of green and purple sulphur bacteria. Until recently, there was little conclusive evidence for the functions of these plasmids, and most were considered to be cryptic. The advent of whole genome sequencing has enabled predictions of possible plasmid functions that can then be tested experimentally. In addition, transcriptomic and proteomic studies in R. sphaeroides 2.4.1 have shown that plasmids play an active role in genome function and may code for essential metabolic processes. This chapter summarises experimental and genomic evidence for plasmid function in the anaerobic anoxygenic phototrophs, with particular emphasis on photosynthetic metabolism, nitrogen oxide reduction, cell wall biosynthesis and heavy metal resistance, and new experimental evidence is presented for the role of the endogenous plasmid in the R. capsulatus strains B10 and SB1003. The evolution of these plasmids will also be considered, and insights from nucleotide sequence comparisons will be combined with those from phylogenetic analysis of plasmid replication modules in the Roseobacter clade of the α- Proteobacteria , which includes aerobic anoxygenic phototrophs.