Jorge M. Santos

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 σs‐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 σs 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 σs 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 σS 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 σS‐directed transcription.

The gene bolA regulates dacA (PBP5), dacC (PBP6) and ampC (AmpC), promoting normal morphology in Escherichia coli

The gene bolA has been shown to trigger the formation of osmotically stable round cells when overexpressed in stationary phase. We show that in poor growth conditions bolA is essential for normal cell morphology in stationary phase and under conditions of starvation. During exponential growth bolA promotes round morphology through a mechanism that is strictly dependent on the two main Escherichia colid,d‐carboxypeptidases, PBP5 and PBP6. The results show that bolA controls the levels of transcription of dacA (PBP5), dacC (PBP6) and ampC (AmpC), a class C β‐lactamase, thus connecting for the first time penicillin binding proteins (PBPs) and β‐lactamases at the level of gene regulation. Furthermore, PBP5 and PBP6 are shown to be independently regulated and to have distinct effects on the peptidoglycan layer. The evidence presented demonstrates that bolA is a regulator of cell wall biosynthetic enzymes with different roles in cell morphology and cell division.

Primers for mating-type diagnosis in Diaporthe and Phomopsis: their use in teleomorph induction in vitro and biological species definition.

Sexual reproduction in ascomycete fungi is governed by the mating-type (MAT) locus. The MAT loci of Diaporthe and its Phomopsis anamorphs differ in only one gene: MAT1-1-1 in mating-type MAT1-1 and MAT1-2-1 in mating-type MAT1-2. In order to diagnose mating-types in Diaporthe and Phomopsis and evaluate their usefulness in teleomorph induction in vitro and biological species delimitation, we designed primers that amplify part of the MAT1-1-1 and MAT1-2-1 genes. MAT phylogenies were generated and compared with ITS and EF1-α phylograms. Species recognised in the EF1-α phylogeny corresponded directly with those determined in the MAT phylogenies. ITS was shown to be highly variable resulting in a large number of phylogenetic species that were discordant with MAT and EF1-α species. Mating experiments were conducted to evaluate the existence of reproductive barriers between some isolates, and their anamorphic morphologies were compared. The primers proved to be useful in the mating-type diagnosis of isolates, selection of compatible mating pairs, and in the assessment of biological species boundaries.

Phylogeny, morphology and pathogenicity of Diaporthe and Phomopsis species on almond in Portugal

The ascomycete genus Diaporthe includes plant pathogens and endophytes on a wide range of hosts including economically important crops. Anamorphs are coelomycetous and reside in the genus Phomopsis. Phomopsis amygdali is the causal agent of twig canker and blight of almonds. In a recent survey of dieback of almonds in Portugal, the most frequent fungi detected were Diaporthe/Phomopsis species. Isolates from almond and other Prunus species were characterised and grouped according to their microsatellite-primed PCR (MSP-PCR) profiles and representatives of the different groups were selected for a phylogenetic study based on the ITS rDNA region (ITS1–5.8S–ITS2). Combining morphological, cultural, molecular and pathogenicity data, three species were distinguished. Phomopsis amygdali was shown to be the main pathogen on almond and is epitypified in the present work. Diaporthe neotheicola is reported for the first time on this host. A third species represented by a single isolate could not be unequivocally identified.

Poly(A)‐polymerase I links transcription with mRNA degradation via σS proteolysis

Bacteria rapidly adapt to changes in growth conditions through control of transcription and specific mRNA degradation. Interplay of both mechanisms must exist in order to achieve fine‐tuned regulation of gene expression. Transcription of the Escherichia coli bolA gene is mediated by the RpoS/σS transcription factor in response to environmental signals. In this report it is shown that the mechanisms of bolA1p mRNA transcription and degradation are tightly connected at the onset of stationary phase and in response to sudden carbon starvation. In stationary phase, bolA1p mRNA levels were reduced 2.5‐fold in a poly(A)‐polymerase I (PAPI) mutant, explained by the significant threefold reduction in σS protein levels in the same strain. Furthermore, fusions with the rpoS gene, analysis of the stability of σS and the levels of RssB indicate that the absence of PAPI enhances RssB‐mediated σS proteolysis specifically in starved cells. The fact that PAPI induces higher cellular levels of a global regulator is a novel finding of wide biological significance. PAPI could work as a linker between transcription and mRNA degradation with the ultimate goal of adapting and surviving to growth‐limiting conditions.

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

Abstract 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.

Denitrifying capabilities of Tetrasphaera and their contribution towards nitrous oxide production in enhanced biological phosphorus removal processes

Denitrifying enhanced biological phosphorus removal (EBPR) systems can be an efficient means of removing phosphate (P) and nitrate (NO3-) with low carbon source and oxygen requirements. Tetrasphaera is one of the most abundant polyphosphate accumulating organisms present in EBPR systems, but their capacity to achieve denitrifying EBPR has not previously been determined. An enriched Tetrasphaera culture, comprising over 80% of the bacterial biovolume was obtained in this work. Despite the denitrification capacity of Tetrasphaera, this culture achieved only low levels of anoxic P-uptake. Batch tests with different combinations of NO3-, nitrite (NO2-) and nitrous oxide (N2O) revealed lower N2O accumulation by Tetrasphaera as compared to Accumulibacter and Competibacter when multiple electron acceptors were added. Electron competition was observed during the addition of multiple nitrogen electron acceptors species, where P uptake appeared to be slightly favoured over glycogen production in these situations. This study increases our understanding of the role of Tetrasphaera-related organisms in denitrifying EBPR systems.