Nitrogen balancing and xylose addition enhances growth capacity and protein content in Chlorella minutissima cultures.

This study aimed to examine the metabolic changes in Chlorella minutissima cells grown under nitrogen-deficient conditions and with the addition of xylose. The cell density, maximum photochemical efficiency, and chlorophyll and lipid levels were measured. The expression of two photosynthetic proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the beta subunit (AtpB) of adenosine triphosphate synthase, were measured. Comparison of cells grown in medium with a 50% reduction in the nitrogen concentration versus the traditional medium solution revealed that the cells grown under nitrogen-deficient conditions exhibited an increased growth rate, higher maximum cell density (12.7×10(6)cellsmL(-1)), optimal PSII efficiency (0.69) and decreased lipid level (25.08%). This study has taken the first steps toward protein detection in Chlorella minutissima, and the results can be used to optimize the culturing of other microalgae.

Bacterial adaptation to cold.

Micro-organisms react to a rapid temperature downshift by triggering a physiological response to ensure survival in unfavourable conditions. Adaptation includes changes in membrane composition and in the translation and transcription machineries. The cold shock response leads to a growth block and overall repression of translation; however, there is the induction of a set of specific proteins that help to tune cell metabolism and readjust it to the new conditions. For a mesophile like E. coli, the adaptation process takes about 4 h. Although the bacterial cold shock response was discovered over two decades ago we are still far from understanding this process. In this review, we aim to describe current knowledge, focusing on the functions of RNA-interacting proteins and RNases involved in cold shock adaptation.

Homologous and heterologous expression of RNase III from Lactococcus lactis.

The endoribonuclease III (RNase III), encoded by the rnc gene, is an important enzyme for RNA metabolism. In this report a chromosomal fragment containing the rnc gene from Lactococcus lactis was cloned and its expression was analyzed. Complementation assays performed in Escherichia coli demonstrate that the lactococcal RNase III (Lac-RNase III) is able to process rRNAs and to regulate the levels of polynucleotide phosphorylase (PNPase). These results demonstrate that the lactococcal enzyme is able to substitute the Ec-RNase III not only in the rRNA processing, but also in the processing of mRNAs. The amount of lactococcal rnc transcript in an E. coli Deltarnc strain was 3.3-fold higher than in the wild type strain, suggesting that the E. coli RNase III triggers the degradation of the heterologous rnc mRNA. Lac-RNase III is able to cleave an in vitro synthesized mRNA substrate specific for the Bacillus subtilis homolog. Using this substrate, we standardized an enzymatic assay which allows the specific detection of the endonucleolytic activity of Lac-RNase III in L. lactis and E. coli crude extracts.

Development of an inducible system to control and easily monitor gene expression in Lactococcus lactis.

This report describes the implementation and use of a maltose-inducible system for regulated gene expression in Lactococcus lactis. The system was established using Green Fluorescent Protein as reporter. The transcription of a gene of interest from the inducible promoter of pLS1RGFP plasmid vector can be easily monitored by fluorescence spectroscopy and microscopy. As an example, the lactococcal ribonuclease III was overproduced in an active form.

RNase II levels change according to the growth conditions: characterization of gmr, a new Escherichia coli gene involved in the modulation of RNase II.

In Escherichia coli, ribonucleases are effectors that rapidly modulate the levels of mRNAs for adaptation to a changing environment. Factors involved in the regulation of these ribonucleases can be relevant for mRNA stability. RNase II is one of the main ribonucleases responsible for exonucleolytic activity in E. coli extracts. We have identified and characterized a new E. coli gene, which was named gmr (gene modulating RNase II). The results demonstrate that a deletion of gmr can be associated with changes in RNase II levels and activity. Western analysis and exoribonuclease activity assays showed a threefold increase in RNase II in the gmr deletion strain. Gmr does not affect RNase II mRNA, but modulates RNase II at the level of protein stability. RNase II protein turnover is slower in the gmr deletion strain. We also show that RNase II levels change in different media, and that this regulation is abolished in a strain lacking gmr. The data presented here show that the regulation of ribonucleolytic activity can depend on growth conditions, and this regulation can be mediated by factors that are not RNases.

Cloning, sequencing and expression of the tetraheme cytochrome c(3) from Desulfovibrio gigas.

The gene encoding the tetraheme cytochrome c(3) from Desulfovibrio gigas was cloned and sequenced from a 2.7-kb EcoRI-PstI insert of D. gigas DNA. The derived amino acid sequence showed that the D. gigas cytochrome c(3) is synthesized as a precursor protein with an N-terminal signal peptide sequence of 25 residues and allowed the correction of the previous reported amino acid sequence (Matias et al. Protein Science 5 (1996) 1342-1354). Expression in D. vulgaris (Hildenborough) was possible by conjugal transfer of a recombinant broad-host-range vector pSUP104 containing a SmaI fragment of the D. gigas cytochrome c(3) gene. Biochemical, immunological and spectroscopic analysis of the purified protein showed that the recombinant cytochrome is identical to that isolated from D. gigas.

RNase II removes the oligo(A) tails that destabilize the rpsO mRNA of Escherichia coli.

Polyadenylation controls mRNA stability in procaryotes, eucaryotes, and organelles. In bacteria, oligo(A) tails synthesized by poly(A) polymerase I are the targets of the 3'-to-5' exoribonucleases: polynucleotide phosphorylase and RNase II. Here we show that RNase II very efficiently removes the oligo(A) tails that can be used as binding sites by PNPase to start degradation of the rpsO mRNA. Both enzymes are impeded by the secondary structure of the transcription terminator at the 3' end of the mRNA. RNase II mostly generates tailless transcripts harboring 2 unpaired nt downstream of the transcription terminator hairpin, whereas PNPase releases molecules that exhibit a single-stranded stretch of 5-7 nt terminated by a tail of 3-5 As. The rpsO mRNAs whose oligo(A) tails have been removed by RNase II are more stable than oligoadenylated molecules that occur in strains deficient for RNase II. Moreover, the rpsO mRNA is stabilized when RNase II is overproduced. This modulation of mRNA stability by RNase II is only observed when poly(A) polymerase I is active. These in vivo data demonstrate that RNase II protects mRNAs ending by stable terminal hairpins, such as primary transcripts, from degradation by poly(A)-dependent ribonucleases.

Degradation of mRNA in bacteria: emergence of ubiquitous features.

The amount of a messenger RNA available for protein synthesis depends on the efficiency of its transcription and stability. The mechanisms of degradation that determine the stability of mRNAs in bacteria have been investigated extensively during the last decade and have begun to be better understood. Several endo- and exoribonucleases involved in the mRNA metabolism have been characterized as well as structural features of mRNA which account for its stability have been determined. The most important recent developments have been the discovery that the degradosome-a multiprotein complex containing an endoribonuclease (RNase E), an exoribonuclease (polynucleotide phosphorylase), and a DEAD box helicase (RhlB)-has a central role in mRNA degradation and that oligo(A) tails synthesized by poly(A) polymerase facilitate the degradation of mRNAs and RNA fragments. Moreover, the phosphorylation status and the base pairing of 5' extremities, together with 3' secondary structures of transcriptional terminators, contribute to the stability of primary transcripts. Degradation of mRNAs can follow several independent pathways. Interestingly, poly(A) tails and multienzyme complexes also control the stability and the degradation of eukaryotic mRNAs. These discoveries have led to the development of refined models of mRNA degradation.

The stationary-phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth.

The Escherichia coli morphogene bolA causes round morphology when overexpressed. The expression of bolA is mainly regulated by a sigmas-dependent gearbox promoter bolA1p. Such regulation results in increased relative levels of expression at slow growth rates, as seen with those attained at the onset of stationary phase. We demonstrate that bolA1p is also induced during early logarithmic growth in response to several forms of stress, and that this induction can be partially sigmas independent. Sudden carbon starvation results in a 17-fold increase in mRNA levels derived from bolA1p 1 h after stress imposition. Increased osmolarity results in a more than 20-fold increase after the same period. Considerable increases in bolA1p mRNA levels were also detected as a result of heat shock, acidic stress and oxidative stress, which has been shown to inhibit sigmas translation. The orders of magnitude of bolA1p induction in log phase due to sudden starvation, osmotic shock and oxidative stress surpass the levels reached in stationary phase. Under sudden carbon starvation and osmotic shock, the cells changed their morphology, resembling those cells in which bolA is overexpressed in stationary phase. Increased expression and morphological changes due to sudden carbon starvation and osmotic shock still occur when sigmaS is not present in a rpoS- background. The results show that expression of bolA is not confined to stationary phase, but it can also play an important role in general stress response. We propose that bolA1p stress induction overrides the normal regulation imposed by growth rate, which is strictly the result of sigmaS-directed transcription.

A comparative analysis of the citrate permease P mRNA stability in Lactococcus lactis biovar diacetylactis and Escherichia coli.

The role of ribonucleases in the control of gene expression remains unknown in lactic acid bacteria. In the present work, we analysed the expression of the citP gene, which encodes the lactococcal citrate permease P, through the stability of the citQRP messenger in both Lactococcus lactis biovar diacetylactis (L. diacetylactis) and Escherichia coli. The chemical half-life for citQRP mRNA observed in L. diacetylactis wild-type strain was abnormally long for bacteria. It was even longer than that detected in E. coli RNase E or RNase III mutant strains. A model of processing and fate of RNA species containing citP gene is presented.

The role of Escherichia coli RNase E and RNase III in the processing of the citQRP operon mRNA from Lactococcus lactis biovar diacetylactis.

Citrate transport in Lactococcus lactis biovar diacetylactis (L. diacetylactis) is catalyzed by citrate permease P (CitP), which is encoded by the plasmidic citP gene. Two partial overlapping open reading frames citQ and citR are located upstream of citP. These two genes, together with citP, constitute the citQRPoperon. In this report it was shown that in L. diacetylactis and Escherichia coli, cit mRNA is subject to the same specific cleavages at a complex secondary structure which includes the central region of citQ and the 5'-end of citR. The role of ribonucleases in the fate of the cit mRNA processing was investigated in E. coli RNase mutant strains. The results obtained indicate that both endoribonucleases RNase E and RNase III are involved in the generation of mRNA processed species. RNase E is responsible for the major cleavages detected within citQ and upstream of citR, whereas RNase III cleaves citR within its ribosomal binding site. Preliminary results indicate the existence of a RNaselll-like enzyme in L. diacetylactis. Based on these results, a model for the role of cit mRNA processing in the expression of citP is presented.

RNA processing is involved in the post-transcriptional control of the citQRP operon from Lactococcus lactis biovar diacetylactis.

The importance of Lactococcus lactis biovar diacetylactis (L. diacetylactis) in the dairy industry is due to its ability to produce aroma compounds, such as acetoin and diacetyl, from citrate. The first step in citrate utilization is its uptake by the cells. In L. diacetylactis, the citrate transport system is encoded by the citQRP operon. We have previously proposed that expression of citQRP operon is regulated at the post-transcriptional level. In this paper, we show that the cit mRNA is processed at a complex secondary structure in L. diacetylactis and Escherichia coli. This secondary structure includes the 5'-terminal two-thirds of citQ and the overlap between citQ and citR. Primer-extension analysis revealed that the major cleavage sites are located upstream of citR and within citQ. In an attempt to identify the enzyme(s) responsible for this cleavage, we have analyzed this processing in E. coli mutants deficient in endoribonucleases. A comparative analysis of cit mRNA degradation was performed in RNase E and RNase III mutants and in wild-type strains using Northern blot hybridization. This analysis revealed that the cit transcript is degraded into several breakdown products, which are significantly stabilized in the mutant lacking RNase III. Our results indicate that the complex secondary structure has a critical role in the control of the expression of cit mRNA. A model for processing is discussed.

Identification and developmental expression of a 5'-3' exoribonuclease from Drosophila melanogaster.

In multicellular organisms, very little is known about the role of mRNA stability in development, and few proteins involved in degradation pathways have been characterized. We have identified the Drosophila homologue of XRN1, which is the major cytoplasmic 5'-3' exoribonuclease in Saccharomyces cerevisiae. The protein sequence of this homologue (pacman) has 59% identity to S. cerevisiae XRN1 and 67% identity to the mouse homologue (mXRN1p) in certain regions. Sequencing of this cDNA revealed that it includes a trinucleotide repeat (CAG)9 which encodes polyglutamine. By directly measuring pacman exoribonuclease activity in yeast, we demonstrate that pacman can complement the yeast XRN1 mutation. Northern blots show a single transcript of approximately 5.2 kb which is abundant only in 0-8-h embryos and in adult males and females. In situ hybridization analysis revealed that the pcm transcripts are maternally derived, and are expressed at high levels in nurse cells. During early embryonic syncytial nuclear divisions, pcm transcripts are homogenously distributed. pcm mRNA is expressed abundantly and ubiquitously throughout the embryo during gastrulation, with high levels in the germ band and head structures. After germ band retraction, pcm transcripts are present at much lower levels, in agreement with the Northern results. Our experiments provide the first example of an exoribonuclease which is differentially expressed throughout development.

Analysis of the in vivo decay of the Escherichia coli dicistronic pyrF-orfF transcript: evidence for multiple degradation pathways.

Messenger RNA decay in Escherichia coli is slowed in pnp-7 (PNPase) rnb-500 (RNase II) rne-1(RNase E) multiple mutants. We have used Northern blots, S1 nuclease protection and primer extension analysis to map 18 endonucleolytic cleavage sites within the pyrF-orfF dicistronic transcript. Although examination of a total of 27 cleavage sites including those determined for the monocistronic trxA transcript revealed a complex pattern, the central four nucleotides within a cluster of 12 residues encompassing the cleavage sites showed a definite A/U preference. Also of interest was the processing of the dicistronic transcript to remove the downstream orfF sequence as a stable but untranslated RNA fragment. The data provide further support for the hypothesis that multiple decay pathways are involved in the decay of a single transcript. In particular, the pyrF-orfF transcript apparently can be degraded either in the 5' to 3' or the 3' to 5' direction. Our results are discussed in light of current models of mRNA decay involving polyadenylation and multiprotein decay complexes.

Determinant role of E. coli RNase III in the decay of both specific and heterologous mRNAs.

A comparative analysis of mRNA decay was carried out in Escherichia coli using the wild-type and an isogenic RNase III deletion strain. We have studied the mRNA degradation from the Escherichia coli gene bolA, the Lactococcus lactis biovar diacetylactis citQRP operon and the Desulfovibrio vulgaris Hildenborough gene cyc. As seen by a dramatic stabilization of the specific mRNAs in the mutant strain, RNase III was crucial for the decay process of these three messages. Since RNase III, unlike RNase E, is not essential for bacterial viability we think that there is potential for using RNase III mutant strains to modulate gene expression.

A new role for RNase II in mRNA decay: striking differences between RNase II mutants and similarities with a strain deficient in RNase E.

The effect of Escherichia coli ribonuclease II and polynucleotide phosphorylase was analysed on the degradation of Desulfovibrio vulgaris cytochrome c3 (cyc) mRNA. In the absence of these exoribonucleolytic activities, cyc mRNA was stabilised but the two enzymes had a different role in its decay. Surprisingly, a temperature-sensitive mutation in ribonuclease II gave a degradation pattern similar to what had been observed in the absence of endoribonuclease E activity. In an RNase II deletion mutant this was not observed. We propose and verify a model in which the temperature-sensitive ribonuclease II interferes with the action of ribonuclease E.

Degradation products of the cytochrome c3 mRNA are similar in Desulfovibrio vulgaris Hildenborough and Escherichia coli.

The transcription and mRNA degradation pattern of a cloned Desulfovibrio vulgaris (Dv) Hildenborough cytochrome c3-encoding gene (cyc) was analyzed in detail, both in Escherichia coli and its native species. Transcription in Dv seems to be controlled by the same promoter elements as in E. coli; the transcription start point (tsp) of this Dv gene has been mapped in both species and found to be identical. A major putative transcription terminator was mapped and it was found to be the same in both organisms. Furthermore, the intermediates in cyc mRNA degradation are similar in both bacterial species.

PNPase modulates RNase II expression in Escherichia coli: implications for mRNA decay and cell metabolism.

PNPase and RNase II are the key regulatory exonucleases controlling mRNA decay in Escherichia coli. The rnb transcripts were found to proceed through the terminator and PNPase was found to be involved in the 3' to 5' degradation of rnb mRNA. Analysis of these longer 3' termini revealed that they are located in UA-rich regions. Comparison of single and double mutants suggested that PNPase and RNase II could have different roles in the degradation of these unstructured regions. We have shown that RNase II levels can vary over a fivefold range in haploid cells and that its expression depends on PNPase levels. PNPase-deficient strains were found to have a 2-2.5-fold increase in RNase II activity, while PNPase-overproducing strains reduced the rnb message and RNase II levels. Conversely, the amount of PNPase in the rnb deletion strain was approximately twofold higher than that in the wild-type strain. These observations suggest that the two main exonucleases are inter-regulated through a fine tuning mechanism. We discuss the implications of these results with regard to mRNA degradation and cell metabolism.

RNase E can inhibit the decay of some degradation intermediates: degradation of Desulfovibrio vulgaris cytochrome c3 mRNA in E coli.

In Escherichia coli, ribonuclease E (RNase E) is a key endonuclease in mRNA decay. We have analysed the role of E coli RNase E on the degradation of a heterologous cytochrome c3 (cyc) mRNA from Desulfovibrio vulgaris Hildenborough. The decay of the cyc transcript in wild-type and mutant E coli cells was followed and the degradation intermediates analysed by Northern blotting and S1 protection analysis. The half-life of total cyc mRNA intermediates was increased in the RNase E mutant. A number of degradation intermediates were stabilised, and new species arose. However, some species decayed faster in the met5 mutant at the non-permissive temperature, suggesting that RNase E might inhibit their degradation. The results indicate that RNase E is involved in cyc mRNA degradation, and, interestingly, decay of certain intermediates could be reduced by this enzyme activity. This may suggest a functional interaction between RNase E and exonucleases, like polynucleotide phosphorylase.

The role of endonucleases in the expression of ribonuclease II in Escherichia coli.

Ribonuclease II (RNase II), encoded by the rnb gene, is one of the two major Escherichia coli exonucleases involved in mRNA degradation. Some of the ribonucleases implicated in this process have recently been shown to be inter-regulated. In this paper we studied the effects of the endonucleases RNase E and RNase III in rnb expression. We have shown that RNase E cleaves the rnb message internally: when this ribonuclease is inactivated rnb mRNA accumulates with a concomitant increase in RNase II activity. RNase III also affects RNase II expression but in an indirect way. We discuss these implications for the regulation of mRNA degradation.