Phenanthrene- and naphthalene-degrading bacteria were isolated from four offshore and nearshore locations in the Gulf of Mexico by using a modified most-probable-number technique. The concentrations of these bacteria ranged from 102 to 106cells per ml of wet surficial sediment in mildly contaminated and noncontaminated sediments. A total of 23 strains of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were obtained. Based on partial 16S ribosomal DNA sequences and phenotypic characteristics, these 23 strains are members of the genus Cycloclasticus. Three representatives were chosen for a complete phylogenetic analysis, which confirmed the close relationship of these isolates to type strain Cycloclasticus pugetii PS-1, which was isolated from Puget Sound. PAH substrate utilization tests which included high-molecular-weight PAHs revealed that these isolates had similar, broad substrate ranges which included naphthalene, substituted naphthalenes, phenanthrene, biphenyl, anthracene, acenaphthene, and fluorene. Degradation of pyrene and fluoranthene occurred only when the strains were incubated with phenanthrene. Two distinct partial PAH dioxygenase iron sulfur protein (ISP) gene sequences were PCR amplified from Puget Sound and Gulf of Mexico Cycloclasticus strains. Phylogenetic analyses of these sequences revealed that one ISP type is related to the bph type of ISP sequences, while the other ISP type is related to the nah type of ISP sequences. The predicted ISP amino acid sequences for the Gulf of Mexico and Puget Sound strains are identical, which supports the hypothesis that these geographically separated isolates are closely related phylogentically.Cycloclasticus species appear to be numerically important and widespread PAH-degrading bacteria in both Puget Sound and the Gulf of Mexico.

Carbon dioxide serves as the preferred electron acceptor during photoheterotrophic growth of nonsulfur purple photosynthetic bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides. This CO2, produced as a result of the oxidation of preferred organic carbon sources, is reduced through reactions of the Calvin-Benson-Bassham reductive pentose phosphate pathway. This pathway is thus crucial to maintain a balanced intracellular oxidation-reduction potential (or redox poise) under photoheterotrophic growth conditions. In the absence of a functional Calvin-Benson-Bassham pathway, either an exogenous electron acceptor, such as dimethylsulfoxide, must be supplied or the organism must somehow develop alternative electron acceptor pathways to preserve the intracellular redox state of the cell. Spontaneous variants of Rba. capsulatus strains deficient in the Calvin-Benson-Bassham pathway that have become photoheterotrophically competent (in the absence of an exogenous electron acceptor) were isolated. These strains (SBP-PHC and RCNd1, RCNd3, and RCNd4) were shown to obviate normal ammonia control and derepress synthesis of the dinitrogenase enzyme complex for the dissipation of excess reducing equivalents and generation of H2 gas via proton reduction. In contrast to previous studies with other organisms, the dinitrogenase reductase polypeptides were maintained in an active and unmodified form in strain SBP-PHC and the respective RCNd strains. Unlike the situation in Rba. sphaeroides, the Rba. capsulatus strains did not regain full ammonia control when complemented with plasmids that reconstituted a functional Calvin-Benson-Bassham pathway. Moreover, dinitrogenase derepression in Rba. capsulatas was responsive to the addition of the auxiliary electron acceptor dimethylsulfoxide. These results indicated a hierarchical control over the removal of reducing equivalents during photoheterotrophic growth that differs from strains of Rba. sphaeroides and Rhodospirillum rubrum deficient in the Calvin-Benson-Bassham pathway.

Various mutant strains were used to examine the regulation and metabolic control of the Calvin-BensonBassham (CBB) reductive pentose phosphate pathway in Rhodobacter capsulatus. Previously, a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient strain (strain SBI/II) was found to show enhanced levels of cbb I and cbb II promoter activities during photoheterotrophic growth in the presence of dimethyl sulfoxide. With this strain as the starting point, additional mutations were made in genes encoding phosphoribulokinase and transketolase and in the gene encoding the LysR-type transcriptional activator, CbbR II . These strains revealed that a product generated by phosphoribulokinase was involved in control of CbbRmediated cbb gene expression in SBI/II. Additionally, heterologous expression experiments indicated that Rhodobacter sphaeroides CbbR responded to the same metabolic signal in R. capsulatus SBI/II and mutant strain backgrounds.The Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway is the primary pathway by which plants, algae, and most photosynthetic bacteria accomplish the fixation of carbon dioxide into organic carbon for subsequent cell metabolism and growth (59). There exist two key enzymes unique to the CBB cycle: ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO), which catalyzes the carboxylation of RuBP, and phosphoribulokinase (PRK), which catalyzes a reaction in which the CO 2 acceptor molecule, RuBP, is generated via the phosphorylation of ribulose 5-phosphate with ATP. CO 2 fixation via the CBB pathway has two major physiological roles in nonsulfur purple photosynthetic bacteria. Under photoautotrophic or chemoautotrophic growth conditions, the key enzymes of the CBB pathway are maximally expressed, with CBB-dependent CO 2 fixation being the major synthetic pathway for the synthesis of organic carbon. Substantially lower levels of CBB cycle enzymes, however, are synthesized under photoheterotrophic growth conditions. Under such conditions, basal levels of CBB pathway enzymes perform the important metabolic function of enabling the cell to employ CO 2 as a preferred electron sink for excess reducing equivalents generated during photoheterotrophic metabolism (58). Thus, when carbon substrates such as L-malate and succinate are metabolized, the CBB cycle facilitates balancing of the oxidation-reduction potential (redox poise) of the cell (17,37,69). Given its different roles in photoheterotrophic and photoautotrophic metabolism, it is not surprising that multiple levels of control influence expression of the CBB system (10,22,46,59,67).Enzymes of the CBB pathway are encoded by the cbb genes, which are organized in regulons in both Rhodobacter capsulatus (44, 45) and Rhodobacter sphaeroides (19,23,24). For both organisms, there are two major cbb operons: the form I (cbb I ) operon, containing form I RubisCO genes (cbbL cbbS), is predominant under autotrophic growth conditions, and the form II (cbb II ) operon, containing the form II RubisCO gene (cbbM), is e...

A transposon mutant of Rhodobacter capsulatus, strain Mal7, that was incapable of photoautotrophic and chemoautotrophic growth and could not grow photoheterotrophically in the absence of an exogenous electron acceptor was isolated. The phenotype of strain Mal7 suggested that the mutation was in some gene(s) not previously shown to be involved in CO(2) fixation control. The site of transposition in strain Mal7 was identified and shown to be in the gene nuoF, which encodes one of the 14 subunits for NADH ubiquinone-oxidoreductase, or complex I. To confirm the role of complex I and nuoF for CO(2)-dependent growth, a site-directed nuoF mutant was constructed (strain SBC1) in wild-type strain SB1003. The complex I-deficient strains Mal7 and SBC1 exhibited identical phenotypes, and the pattern of CO(2) fixation control through the Calvin-Benson-Bassham pathway was the same for both strains. It addition, it was shown that electron transport through complex I led to differential control of the two major cbb operons of this organism. Complex I was further shown to be linked to the control of nitrogen metabolism during anaerobic photosynthetic growth of R. capsulatus.