Interacting regulatory networks in the facultative photosynthetic bacterium, Rhodobacter sphaeroides 2.4.1.

Regulation of photosynthetic membrane synthesis in Rhodobacter sphaeroides 2.4.1 is dependent on the interactions of numerous regulatory elements, with two of the most important being the cbb(3) terminal oxidase and the PrrBAC two-component regulatory system. Here, we reveal that the cbb(3) terminal oxidase possesses extensive, additional regulatory activities under anaerobic conditions, and that the PrrBAC system is further involved in the regulation of the expression of more than 20% of the R. sphaeroides genome under anaerobic conditions, extending well beyond functions related to redox gene expression.

Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase.

Two new loci, prrB and prrC, involved in the positive regulation of photosynthesis gene expression in response to anaerobiosis, have been identified in Rhodobacter sphaeroides. prrB encodes a sensor histidine kinase that is responsive to the removal of oxygen and functions through the response regulator PrrA. Inactivation of prrB results in a substantial reduction of photosynthetic spectral complexes as well as in the inability of cells to grow photosynthetically at low to medium light intensities. Together, prrB and prrA provide the major signal involved in synthesis of the specialized intracytoplasmic membrane (ICM), harboring components essential to the light reactions of photosynthesis. Previously, J. K. Lee and S. Kaplan (J. Bacteriol. 174:1158-1171, 1992) identified a mutant which resulted in high-level expression of the puc operon, encoding the apoproteins giving rise to the B800-850 spectral complex, in the presence of oxygen as well as in the synthesis of the ICM under conditions of high oxygenation. This mutation is shown to reside in prrB, resulting in a leucine-to-proline change at position 78 in mutant PrrB (PRRB78). Measurements of mRNA levels in cells containing the prrB78 mutation support the idea that prrB is a global regulator of photosynthesis gene expression. Two additional mutants, PRRB1 and PRRB2, which make two truncated forms of the PrrB protein, possess substantially reduced amounts of spectral complexes. Although the precise role of prrC remains to be determined, evidence suggests that it too is involved in the regulatory cascade involving prrB and prrA. The genetic organization of the photosynthesis response regulatory (PRR) region is discussed.

Anaerobic control of colicin E1 production.

Expression of the cea gene, which is carried by the ColE1 plasmid and which encodes colicin E1, was found to be greatly increased when the cells were grown anaerobically. By using cea-lacZ fusions to quantitate expression, aerobic levels were found to be only a few percent of the anaerobic levels. The anaerobic increase in expression was observed both in protein and in operon fusions, indicating that its regulation occurred at the level of transcription. It was also found to require a functional fnr gene and to occur when the cea-lacZ fusion was present as a single copy in the bacterial chromosome instead of in the multicopy ColE1 plasmid. Anaerobic expression was regulated by the SOS response and catabolite repression as is aerobic expression. The start site of the mRNA produced under anaerobic conditions was mapped by primer extension and found to be the same as the start for mRNA produced under aerobic conditions. These observations show that the cea gene is anaerobically regulated and that the Fnr protein is a positive regulator of transcription of this gene.

prrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides.

A new locus, prrA, involved in the regulation of photosynthesis gene expression in response to oxygen, has been identified in Rhodobacter sphaeroides. Inactivation of prrA results in the absence of photosynthetic spectral complexes. The prrA gene product has strong homology to response regulators associated with signal transduction in other prokaryotes. When prrA is present in multiple copies, cells produce light-harvesting complexes under aerobic growth conditions, suggesting that prrA affects photosynthesis gene expression positively in response to oxygen deprivation. Analysis of the expression of puc::lacZ fusions in wild-type and PrrA- cells revealed a substantial decrease in LacZ expression in the absence of prrA under all conditions of growth, especially when cells were grown anaerobically in the dark in the presence of dimethyl sulfoxide. Northern (RNA) and slot blot hybridizations confirmed the beta-galactoside results for puc and revealed additional positive regulation of puf, puhA, and cycA by PrrA. The effect of truncated PrrA on photosynthesis gene expression in the presence of low oxygen levels can be explained by assuming that PrrA may be effective as a multimer. PrrA was found to act on the downstream regulatory sequences (J. K. Lee and S. Kaplan, J. Bacteriol. 174:1146-1157, 1992) of the puc operon regulatory region. Finally, two spontaneous prrA mutations that abolish prrA function by changing amino acids in the amino-terminal domain of the protein were isolated.

Complex regulatory activities associated with the histidine kinase PrrB in expression of photosynthesis genes in Rhodobacter sphaeroides 2.4.1.

Rhodobacter sphaeroides 2.4.1 synthesizes a specialized photosynthetic membrane upon reduction of the O2 tension below threshold levels. The genes prrB and prrA encode a sensor kinase and a response regulator, respectively, of a two-component regulatory system that presumably is involved in transduction of the signal(s) that monitors alterations in oxygen levels. A third gene, prrC, is also involved in this cascade of events. Previously, we described a mutant form of PrrB, namely, PrrB78 (J. M. Eraso and S. Kaplan, J. Bacteriol. 177:2695-2706, 1995), which results in aerobic expression of the photosynthetic apparatus. Here we examine three mutated forms of the prrB gene that have the potential to encode truncated polypeptides containing the N-terminal 6, 63, or 163 amino acids, respectively. The resulting mutant strains showed residual levels of the light-harvesting spectral complexes and had diminished photosynthetic growth rates at high light intensities with no discernible growth under intermediate or low light conditions. When either lacZ transcriptional fusions or direct mRNA determinations were used to monitor specific photosynthesis gene expression, all the mutant strains showed unexpectedly high levels of gene expression when compared to mutant strains affected in prrA. Conversely, when translational fusions were used to monitor photosynthesis gene expression in these mutant strains, expression of both puc and puf operons was reduced, especially puf expression. In light of these studies and those of the PrrB78 mutant, the data suggest that PrrA can be activated in situ by something other than PrrB, and it also appears that PrrB can function as a negative regulator acting through PrrA. Finally, we consider the role of the Prr regulatory system in the posttranscriptional control of photosynthesis gene expression.

From redox flow to gene regulation: role of the PrrC protein of Rhodobacter sphaeroides 2.4.1.

Activation of photosynthesis (PS) gene expression by the PrrBA two-component activation system in Rhodobacter sphaeroides 2.4.1 results from the interruption of an inhibitory signal originating from the cbb(3) cytochrome c oxidase via its interaction with oxygen, in conjunction with the Rdx redox proteins. The CcoQ protein, encoded by the ccoNOQP operon, which encodes the cbb(3) cytochrome c oxidase, was shown to act as a "transponder" that conveys the signal derived from reductant flow through cbb(3) to oxygen, to the Prr system. To further define the elements comprising this signal transduction pathway we considered the prrC gene product, which to date possessed no definable role in this signal transduction pathway despite its being part of the prrBCA gene cluster. Similar to mutations in cbb(3) and rdx, suitably constructed prrC deletion mutations lead to PS gene expression in the presence of high oxygen. Unlike mutations that remove cbb(3) terminal oxidase activity or Rdx function, the PrrC deletion mutant shows no effect upon cbb(3) activity, nor does it affect the ratio of the carotenoid (Crt) spheroidene (SE) to spheroidenone (SO). Thus, the PrrC deletion mutant behaves identically to the CcoQ deletion mutant. Taking these and previous results together, we suggest that PrrC is located upstream of the two-component PrrBA activation system in the signal transduction pathway but downstream of the cbb(3) cytochrome c oxidase and its "transponder" CcoQ. The PrrC deletion mutant was also shown to lead to an increase in the DorA protein under aerobic conditions as was shown earlier for the cbb(3) mutant. Finally, PrrC is a member of a highly conserved family of proteins found in both prokaryotes and eukaryotes, and this appears to be the first instance in which a direct regulatory role has been ascribed to a member of this protein family.

Interacting regulatory circuits involved in orderly control of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

FnrL, the homolog of the global anaerobic regulator Fnr, is required for the induction of the photosynthetic apparatus in Rhodobacter sphaeroides 2.4.1. Thus, the precise role of FnrL in photosynthesis (PS) gene expression and its interaction(s) with other regulators of PS gene expression are of considerable importance to our understanding of the regulatory circuitry governing spectral complex formation. Using a CcoP and FnrL double mutant strain, we obtained results which suggested that FnrL is not involved in the transduction of the inhibitory signal, by which PS gene expression is "silenced," emanating from the cbb(3) oxidase encoded by the ccoNOQP operon under aerobic conditions. The dominant effect of the ccoP mutation in the FnrL mutant strain with respect to spectral complex formation under aerobic conditions and restoration of a PS-positive phenotype suggested that inactivation of the cbb(3) oxidase to some extent bypasses the requirement for FnrL in the formation of spectral complexes. Additional analyses revealed that anaerobic induction of the bchE, hemN, and hemZ genes, which are involved in the tetrapyrrole biosynthetic pathways, requires FnrL. Thus, FnrL appears to be involved at multiple loci involved in the regulation of PS gene expression. Additionally, bchE was also shown to be regulated by the PrrBA two-component system, in conjunction with hemN and hemZ. These and other results to be discussed permit us to more accurately describe the role of FnrL as well as the interactions between the FnrL, PrrBA, and other regulatory circuits in the regulation of PS gene expression.

A redox-responsive pathway for aerobic regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

To further understand the proposed signal transduction pathway involving the presumed redox proteins RdxBH and cbb3 cytochrome oxidase in Rhodobacter sphaeroides 2.4.1, a series of mutants lacking components of both the Prr two-component activation system and the cbb3-type cytochrome oxidase or RdxBH were constructed. We report that under highly aerobic conditions, aberrant photosynthesis gene expression and spectral complex formation typical of cbb3- or RdxBH-deficient mutants were no longer observed when either prrA (encoding the response regulator of the Prr system) or prrB (encoding the presumed sensor kinase) was also deleted. These double-mutant strains are phenotypically identical to single-mutant PrrA and PrrB strains, suggesting that the signal(s) originating from the cbb3 terminal oxidase affects downstream puc and puf operon expression by acting exclusively through the Prr system. When the same double-mutant strains were examined under anaerobic dark dimethyl sulfoxide growth conditions, photosynthesis gene expression was obligatorily linked to the two-component activation system. However, photosynthesis gene expression under the same growth conditions was significantly higher in the cbb3 mutant strain when compared to that in the wild type. Similarly, under anaerobic photosynthetic conditions the high levels of the oxidized carotenoid, spheroidenone, which accumulate in cbb3-deficient mutants were nearly restored to normal in a PrrB- CcoP- double mutant. This observation, together with previously published results, suggests that the regulation of the CrtA-catalyzed reaction possesses both transcriptional and posttranscriptional regulatory effectors. We propose that the cbb3 cytochrome oxidase, which by definition can interact with external oxygen, serves to control the activity of the Prr two-component activation system under both aerobic and anaerobic conditions. Although independent from the cbb3 oxidase, the RdxBH proteins are also required for normal functioning of the Prr two-component activation system and are therefore believed to lie between the cbb3 oxidase in this oxygen-sensing, redox signaling pathway and the Prr activation system.

Differential carotenoid composition of the B875 and B800-850 photosynthetic antenna complexes in Rhodobacter sphaeroides 2.4.1: involvement of spheroidene and spheroidenone in adaptation to changes in light intensity and oxygen availability.

Rhodobacter sphaeroides 2.4.1 is a member of the nonsulfur purple facultative photosynthetic proteobacteria, capable of growth under a variety of cultivation conditions. In addition to the structural polypeptides and bacteriochlorophyll, the two major antenna complexes, B875 and B800-850, contain a variety of carotenoids which are an important structural and functional component of the membrane-bound photosynthetic complexes of this bacterium. Two major carotenoids, spheroidene and its keto derivative, spheroidenone, are differentially synthesized by R. sphaeroides, depending on the growth conditions. Spheroidene prevails during growth under anaerobic conditions and low light intensities, whereas spheroidenone is predominant in semiaerobically grown cells or during anaerobic growth at high light intensities. In this study, we demonstrate that in wild-type cells, spheroidene is predominantly associated with the B800-850 photosynthetic antenna complex and spheroidenone is more abundant in the B875 complex. Exploiting mutants defective in the biosynthesis of either the B875 or B800-850 light-harvesting complex, we demonstrate an association between the formation of either the B875 or B800-850 complex, on the one hand, and the accumulation of spheroidenone or spheroidene, on the other. The possible involvement of the conversion of spheroidene to spheroidenone as a significant control mechanism involved in the adaptation of R. sphaeroides to changes in light intensity and oxygen tension is discussed.

Increased production of colicin E1 in stationary phase.

The synthesis of colicin E1 is known to be regulated by the SOS response, anaerobiosis, and catabolite repression. The expression of cea-lacZ fusions was also found to be stimulated when cells reached stationary phase. This increase in expression was determined to be due to depletion of nutrients from the medium, since the addition of fresh medium reversed the effect. Expression of the fusion increased when cells were starved in 10 mM MgSO4 and when they were grown in conditioned medium in which cells had been grown previously. The stimulation of expression occurred when the cea-lacZ fusion was present in single-copy as well as in multicopy plasmids. Finally, the data were consistent with this increase being independent of the SOS response, anaerobiosis, catabolite repression, and integration host factor as well as the stationary-phase regulators encoded by rpoS and lrp.

Transcriptional regulation of puc operon expression in Rhodobacter sphaeroides. Involvement of an integration host factor-binding sequence.

The putative overlapping consensus sequences (-129 to -105) for binding of fumarate nitrate reductase regulator- and integration host factor (IHF)-like proteins to puc operon upstream DNA of Rhodobacter sphaeroides was protected from DNase I digestion by purified Escherichia coli IHF. The binding of E. coli IHF to the purported IHF-binding site in the puc upstream DNA is highly sequence-specific. The recorded binding affinity was significantly lower than that of E. coli IHF to the lambda attP site. Employing site-directed changes in the DNA sequence within the -129 to -105 region, a loss in IHF binding, as monitored through gel retardation analysis, was correlated with alterations in puc operon expression monitored through the use of puc::lacZ transcriptional fusions. These results suggest that the IHF-binding site is involved in repression of puc operon transcription by oxygen as well as modulation of puc operon transcription levels by incident light intensity. Mutations specific to the upstream half of the putative fumarate nitrate reductase regulator-binding site of the puc upstream DNA did not show any physiological effects under the experimental conditions employed. Taken together, these studies reveal that the DNA sequence between -129 to -105 may involve facilitation of the interaction between upstream and downstream cis-acting regulatory sequences involved in puc operon expression.

[Renal echographic volumetry in the child. Normal patterns]. / Volumetría ecográfica renal en el niño. I. Patrones normales.

After experimental verification of an excellent correlation between volume measurements by water displacement and echography we have checked that there were no differences among several renal measurements performed in children by the same person. In our normal population of 313 children we assumed that the best correlation between somatometric and renal measurements parameter was between weight and renal volume. We have developed volumetric growth charts for both kidneys. There were no significant difference between right and left kidney.