Staphylococcus aureus is an opportunistic bacterium of the human body and a leading cause of nosocomial infections. Methicillin resistant S. aureus (MRSA) infections involving biofilm lead to higher mortality and morbidity in patients. Biofilm causes serious clinical issues, as it mitigates entry of antimicrobials to reach the etiological agents. It plays an important role in resilient chronic infections which place an unnecessary burden on antibiotics and the associated costs. To combat drug-resistant infection involving biofilm, there is a need to discover potential anti-biofilm agents. In this study, activity of polyphenolic flavonoid glabridin against biofilm formation of methicillin resistant clinical isolates of S. aureus is being reported for the first time. Crystal violet assay and scanning electron microscopy evidences shows that glabridin prevents formation of cells clusters and attachment of methicillin resistant clinical isolate (MRSA 4423) of S. aureus to the surface in a dose dependent manner. Gel free proteomic analysis of biofilm matrix by LC-ESI-QTOF confirmed the existence of several proteins known to be involved in cells adhesion. Furthermore, expression analysis of cell surface proteins revealed that glabridin significantly down regulates an abundance of several surface-associated adhesins including fibronectin binding proteins (FnbA, FnbB), serine-aspartate repeat-containing protein D (SdrD), immunoglobulin-binding protein G (Sbi), and other virulence factors which were induced by extracellular glucose in MRSA 4423. In addition, several moonlighting proteins (proteins with multiple functions) such as translation elongation factors (EF-Tu, EF-G), chaperone protein (DnaK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) were detected on the cell surface wherein their abundance was inversely proportional to surface-associated adhesins. This study clearly suggests that glabridin prevents biofilm formation in S. aureus through modulation of the cell surface proteins.

Bacteria belonging to the Alphaproteobacteria normally harbour multiple copies of the heat shock sigma factor (known as σ³², σ^H or RpoH). Azospirillum brasilense, a non-photosynthetic rhizobacterium, harbours five copies of rpoH genes, one of which is an rpoH2 homologue. The genes around the rpoH2 locus in A. brasilense show synteny with that found in rhizobia. The rpoH2 of A. brasilense was able to complement the temperature-sensitive phenotype of the Escherichia coli rpoH mutant. Inactivation of rpoH2 in A. brasilense results in increased sensitivity to methylene blue and to triphenyl tetrazolium chloride (TTC). Exposure of A. brasilense to TTC and the singlet oxygen-generating agent methylene blue induced several-fold higher expression of rpoH2. Comparison of the proteome of A. brasilense with its rpoH2 deletion mutant and with an A. brasilense strain overexpressing rpoH2 revealed chaperone GroEL, elongation factors (Ef-Tu and EF-G), peptidyl prolyl isomerase, and peptide methionine sulfoxide reductase as the major proteins whose expression was controlled by RpoH2. Here, we show that the RpoH2 sigma factor-controlled photooxidative stress response in A. brasilense is similar to that in the photosynthetic bacterium Rhodobacter sphaeroides, but that RpoH2 is not involved in the detoxification of methylglyoxal in A. brasilense.

The bacterium Paracoccus denitrificans is a model for the process of denitrification, by which nitrate is reduced to dinitrogen during anaerobic growth. Denitrification is important for soil fertility and greenhouse gas emission and in waste and water treatment processes. The ability of bacteria to grow as a biofilm attached to a solid surface is important in many different contexts. In this paper, we report that attached growth of P. denitrificans is stimulated by nitric oxide, an intermediate in the denitrification pathway. We also show that calcium ions stimulate attached growth, and we identify a large calcium binding protein that is required for growth on a polystyrene surface. We identify components of a signaling pathway through which nitric oxide may regulate biofilm formation. Our results point to an intimate link between metabolic processes and the ability of P. denitrificans to grow attached to a surface.

Azospirillum brasilense, a plant-growth-promoting rhizobacterium, is exposed to changes in its abiotic environment, including fluctuations in temperature, salinity, osmolarity, oxygen concentration and nutrient concentration, in the rhizosphere and in the soil. Since extra-cytoplasmic function (ECF) sigma factors play an important role in stress adaptation, we analysed the role of ECF sigma factor (also known as RpoE or σ ^E) in abiotic stress tolerance in A. brasilense. An in-frame rpoE deletion mutant of A. brasilense Sp7 was carotenoidless and slow-growing, and was sensitive to salt, ethanol and methylene blue stress. Expression of rpoE in the rpoE deletion mutant complemented the defects in growth, carotenoid biosynthesis and sensitivity to different stresses. Based on data from reverse transcriptase-PCR, a two-hybrid assay and a pull-down assay, we present evidence that rpoE is cotranscribed with chrR and the proteins synthesized from these two overlapping genes interact with each other. Identification of the transcription start site by 5′ rapid amplification of cDNA ends showed that the rpoE–chrR operon was transcribed by two promoters. The proximal promoter was less active than the distal promoter, whose consensus sequence was characteristic of RpoE-dependent promoters found in alphaproteobacteria. Whereas the proximal promoter was RpoE-independent and constitutively expressed, the distal promoter was RpoE-dependent and strongly induced in response to stationary phase and elevated levels of ethanol, salt, heat and methylene blue. This study shows the involvement of RpoE in controlling carotenoid synthesis as well as in tolerance to some abiotic stresses in A. brasilense, which might be critical in the adaptation, survival and proliferation of this rhizobacterium in the soil and rhizosphere under stressful conditions.

Extracytoplasmic function (ECF) sigma factors (σE) are known to bring about changes in gene expression to enable bacteria to adapt to different stresses. The Azospirillum brasilense Sp245 genome harbours nine genes encoding σE, of which two are adjacent to the genes encoding ChrR-type zinc-binding anti-sigma (ZAS) factors. We describe here the role and regulation of a new pair of rpoE–chrR, which was found in the genome of A. brasilense Sp7 in addition to the previously described rpoE–chrR pair (designated rpoE1–chrR1). The rpoE2–chrR2 pair is also cotranscribed, and their products show protein–protein interaction. The −10 and −35 promoter elements of rpoE2–chrR2 and rpoE1–chrR1 were similar but not identical. Unlike the promoter of rpoE1–chrR1, the rpoE2–chrR2 promoter was neither autoregulated nor induced by oxidative stress. Inactivation of chrR2 or overexpression of rpoE2 in A. brasilense Sp7 resulted in an overproduction of carotenoids. It also conferred resistance to oxidative stresses and antibiotics. By controlling the synthesis of carotenoids, initiation and elongation of translation, protein folding and purine biosynthesis, RpoE2 seems to play a crucial role in preventing and repairing the cellular damage caused by oxidative stress. Lack of autoregulation and constitutive expression of rpoE2–chrR2 suggest that RpoE2–ChrR2 may provide a rapid mechanism to cope with oxidative stress, wherein singlet oxygen (1O2)-mediated dissociation of the RpoE2–ChrR2 complex might release RpoE2 to drive the expression of its target genes.

Bacteria must acquire the essential element zinc from extremely limited environments, and this function is performed largely by ATP binding cassette (ABC) transporters. These systems rely on a periplasmic or extracellular solute binding protein (SBP) to bind zinc specifically and with high affinity and deliver it to the membrane permease for import into the cytoplasm. However, zinc acquisition systems in bacteria may be more complex, involving multiple transporters and other periplasmic or extracellular zinc binding proteins. Here we describe the zinc acquisition functions of two zinc SBPs (ZnuA and AztC) and a novel periplasmic metallochaperone (AztD) in Paracoccus denitrificans. ZnuA was characterized in vitro and demonstrated to bind as many as 5 zinc ions with high affinity. It does not interact with AztD, in contrast to what has been demonstrated for AztC, which is able to acquire a single zinc ion through associative transfer from AztD. Deletions of the corresponding genes singly and in combination show that either AztC or ZnuA is sufficient and essential for robust growth in zinc limited media. Although AztD cannot support transport of zinc into the cytoplasm, it likely functions to store zinc in the periplasm for transfer through the AztABCD system.