DNA recognition and transcriptional regulation by the WhiA sporulation factor

Sporulation in the filamentous bacteria Streptomyces coelicolor is a tightly regulated process involving aerial hyphae growth, chromosome segregation, septation and spore maturation. Genetic studies have identified numerous genes that regulate sporulation, including WhiA and the sigma factor WhiG. WhiA, which has been postulated to be a transcriptional regulator, contains two regions typically associated with DNA binding: an N-terminal domain similar to LAGLIDADG homing endonucleases, and a C-terminal helix-turn-helix domain. We characterized several in vitro activities displayed by WhiA. It binds at least two sporulation-specific promoters: its own and that of parABp2. DNA binding is primarily driven by its HTH domain, but requires full-length protein for maximum affinity. WhiA transcription is stimulated by WhiG, while the WhiA protein binds directly to WhiG (leading to inhibition of WhiG-dependent transcription). These separate activities, which resemble a possible feedback loop, may help coordinate the closely timed cessation of aerial growth and subsequent spore formation

A novel compartment, the 'subqpical stem' of the aerial hyphae, is the location of a sigN-dependent, developmentally distinct transcription in Streptomyces coelicolor.

Streptomyces coelicolor has nine SigB-like RNA polymerase sigma factors, several of them implicated in morphological differentiation and/or responses to different stresses. One of the nine, SigN, is the focus of this article. A constructed sigN null mutant was delayed in development and exhibited a bald phenotype when grown on minimal medium containing glucose as carbon source. One of two distinct sigN promoters, sigNP1, was active only during growth on solid medium, when its activation coincided with aerial hyphae formation. Transcription from sigNP1 was readily detected in several whi mutants (interrupted in morphogenesis of aerial mycelium into spores), but was absent from all bld mutants tested, suggesting that sigNP1 activity was restricted to the aerial hyphae. It also depended on sigN, thus sigN was autoregulated. Mutational and transcription studies revealed no functional significance to the location of sigN next to sigF, encoding another SigB-like sigma factor. We identified another potential SigN target, nepA, encoding a putative small secreted protein. Transcription of nepA originated from a single, aerial hyphae-specific and sigN-dependent promoter. While in vitro run-off transcription using purified SigN on the Bacillus subtilis ctc promoter confirmed that SigN is an RNA polymerase sigma factor, SigN failed to initiate transcription from sigNP1 and from the nepA promoter in vitro. Additional in vivo data indicated that further nepA upstream sequences, which are likely to bind a potential activator, are required for successful transcription. Using a nepA–egfp transcriptional fusion we located nepA transcription to a novel compartment, the ‘subapical stem’ of the aerial hyphae. We suggest that this newly recognized compartment defines an interface between the aerial and vegetative parts of the Streptomyces colony and might also be involved in communication between these two compartments

The dpsA Gene of Streptomyces coelicolor: Induction of Expression from a Single Promoter in Response to Environmental Stress or during Development

The DpsA protein plays a dual role in Streptomyces coelicolor, both as part of the stress response and contributing to nucleoid condensation during sporulation. Promoter mapping experiments indicated that dpsA is transcribed from a single, sigB-like dependent promoter. Expression studies implicate SigH and SigB as the sigma factors responsible for dpsA expression while the contribution of other SigB-like factors is indirect by means of controlling sigH expression. The promoter is massively induced in response to osmotic stress, in part due to its sensitivity to changes in DNA supercoiling. In addition, we determined that WhiB is required for dpsA expression, particularly during development. Gel retardation experiments revealed direct interaction between apoWhiB and the dpsA promoter region, providing the first evidence for a direct WhiB target in S. coelicolor

A trehalose biosynthetic enzyme doubles as an osmotic stress sensor to regulate bacterial morphogenesis

The dissacharide trehalose is an important intracellular osmoprotectant and the OtsA/B pathway is the principal pathway for trehalose biosynthesis in a wide range of bacterial species. Scaffolding proteins and other cytoskeletal elements play an essential role in morphogenetic processes in bacteria. Here we describe how OtsA, in addition to its role in trehalose biosynthesis, functions as an osmotic stress sensor to regulate cell morphology in Arthrobacter strain A3. In response to osmotic stress, this and other Arthrobacter species undergo a transition from bacillary to myceloid growth. An otsA null mutant exhibits constitutive myceloid growth. Osmotic stress leads to a depletion of trehalose-6-phosphate, the product of the OtsA enzyme, and experimental depletion of this metabolite also leads to constitutive myceloid growth independent of OtsA function. In vitro analyses indicate that OtsA can self-assemble into protein networks, promoted by trehalose-6-phosphate, a property that is not shared by the equivalent enzyme from E. coli, despite the latter's enzymatic activity when expressed in Arthrobacter. This, and the localization of the protein in non-stressed cells at the mid-cell and poles, indicates that OtsA from Arthrobacter likely functions as a cytoskeletal element regulating cell morphology. Recruiting a biosynthetic enzyme for this morphogenetic function represents an intriguing adaptation in bacteria that can survive in extreme environments

Glucose Starvation Boosts Entamoeba histolytica Virulence

The unicellular parasite, Entamoeba histolytica, is exposed to numerous adverse conditions, such as nutrient deprivation, during its life cycle stages in the human host. In the present study, we examined whether the parasite virulence could be influenced by glucose starvation (GS). The migratory behaviour of the parasite and its capability to kill mammalian cells and to lyse erythrocytes is strongly enhanced following GS. In order to gain insights into the mechanism underlying the GS boosting effects on virulence, we analyzed differences in protein expression levels in control and glucose-starved trophozoites, by quantitative proteomic analysis. We observed that upstream regulatory element 3-binding protein (URE3-BP), a transcription factor that modulates E.histolytica virulence, and the lysine-rich protein 1 (KRiP1) which is induced during liver abscess development, are upregulated by GS. We also analyzed E. histolytica membrane fractions and noticed that the Gal/GalNAc lectin light subunit LgL1 is up-regulated by GS. Surprisingly, amoebapore A (Ap-A) and cysteine proteinase A5 (CP-A5), two important E. histolytica virulence factors, were strongly down-regulated by GS. While the boosting effect of GS on E. histolytica virulence was conserved in strains silenced for Ap-A and CP-A5, it was lost in LgL1 and in KRiP1 down-regulated strains. These data emphasize the unexpected role of GS in the modulation of E.histolytica virulence and the involvement of KRiP1 and Lgl1 in this phenomenon

One of the Two Genes Encoding Nucleoid-Associated HU Proteins in Streptomyces coelicolor Is Developmentally Regulated and Specifically Involved in Spore Maturation

Streptomyces genomes encode two homologs of the nucleoid-associated HU proteins. One of them, here designated HupA, is of a conventional type similar to E. coli HU and HU , while the other, HupS, is a two-domain protein. In addition to the N-terminal part that is similar to that of HU proteins, it has a C-terminal domain that is similar to the alanine- and lysine-rich C termini of eukaryotic linker histones. Such two-domain HU proteins are found only among Actinobacteria. In this phylum some organisms have only a single HU protein of the type with a C-terminal histone H1-like domain (e.g., Hlp in Mycobacterium smegmatis), while others have only a single conventional HU. Yet others, including the streptomycetes, produce both types of HU proteins. We show here that the two HU genes in Streptomyces coelicolor are differentially regulated and that hupS is specifically expressed during sporulation, while hupA is expressed in vegetative hyphae. The developmental upregulation of hupS occurred in sporogenic aerial hyphal compartments and was dependent on the developmental regulators whiA, whiG, and whiI. HupS was found to be nucleoid associated in spores, and a hupS deletion mutant had an average nucleoid size in spores larger than that in the parent strain. The mutant spores were also defective in heat resistance and spore pigmentation, although they possessed apparently normal spore walls and displayed no increased sensitivity to detergents. Overall, the results show that HupS is specifically involved in sporulation and may affect nucleoid architecture and protection in spores of S. coelicolor

SmeA, a small membrane protein with multiple functions in Streptomyces sporulation including targeting of a SpoIIIE/FtsK-like protein to cell division septa

Sporulation in aerial hyphae of Streptomyces coelicolor involves profound changes in regulation of fundamental morphogenetic and cell cycle processes to convert the filamentous and multinucleoid cells to small unigenomic spores. Here, a novel sporulation locus consisting of smeA (encoding a small putative membrane protein) and sffA (encoding a SpoIIIE/FtsK-family protein) is characterized. Deletion of smeA-sffA gave rise to pleiotropic effects on spore maturation, and influenced the segregation of chromosomes and placement of septa during sporulation. Both smeA and sffA were expressed specifically in apical cells of sporogenic aerial hyphae simultaneously with or slightly after Z-ring assembly. The presence of smeA-like genes in streptomycete chromosomes, plasmids and transposons, often paired with a gene for a SpoIIIE/FtsK- or Tra-like protein, indicates that SmeA and SffA functions might be related to DNA transfer. During spore development SffA accumulated specifically at sporulation septa where it colocalized with FtsK. However, sffA did not show redundancy with ftsK, and SffA function appeared distinct from the DNA translocase activity displayed by FtsK during closure of sporulation septa. The septal localization of SffA was dependent on SmeA, suggesting that SmeA may act as an assembly factor for SffA and possibly other proteins required during spore maturation

Bacterial growth kinetics estimation by fluorescence in situ hybridization and spectrofluorometric quantification

Aims: The aim of this study was to develop a specific and rapid method to identify and quantify relevant bacterial populations in mixed biomass by spectrofluorometric quantification (SQ) of whole cells hybridized with fluorescently labelled oligonucleotide probes targeting mature 16S ribosomal RNA (rRNA). Probe targeting the precursor of rRNA synthesis was also employed because it was being suggested as more indicative of the activity state of the micro-organisms. Methods and Results: Original fluorescence in situ hybridization protocol was modified to be applied to liquid samples and the fluorescence emission from the Cy3-labelled cells was measured by spectrofluorometry. The method was calibrated on an exponentially growing cell suspension of Acinetobacter johnsonii and was successfully applied to generate kinetic data. No substantial difference in the estimated maximum specific growth rate (mu(max)) values was found between the SQ method and the classical method, using absorbance at 420 nm (6.2 d(-1)). The preliminary validation tests showed their direct applicability to target enriched cultures. Conclusions: This study demonstrated the validity of the SQ method to easily quantify the concentration and to determine the growth rate of specific micro-organisms present in mixed cultures. Significan ce and Impact of the Study: The proposed method can be directly utilized for quantification and kinetic characterization of microbial enrichments. It has the advantage of being easily applicable using simple, inexpensive equipment suitable for routine analysis

MreB of Streptomyces coelicolor is not essential for vegetative growth but is required for the integrity of aerial hyphae and spores

Microbial Biotechnolog