An ancient bacterial zinc acquisition system identified from a cyanobacterial exoproteome.

Bacteria have developed fine-tuned responses to cope with potential zinc limitation. The Zur protein is a key player in coordinating this response in most species. Comparative proteomics conducted on the cyanobacterium Anabaena highlighted the more abundant proteins in a zur mutant compared to the wild type. Experimental evidence showed that the exoprotein ZepA mediates zinc uptake. Genomic context of the zepA gene and protein structure prediction provided additional insights on the regulation and putative function of ZepA homologs. Phylogenetic analysis suggests that ZepA represents a primordial system for zinc acquisition that has been conserved for billions of years in a handful of species from distant bacterial lineages. Furthermore, these results show that Zur may have been one of the first regulators of the FUR family to evolve, consistent with the scarcity of zinc in the ecosystems of the Archean eon.

SepT, a novel protein specific to multicellular cyanobacteria, influences peptidoglycan growth and septal nanopore formation in Anabaena sp. PCC 7120.

IMPORTANCE: Multicellular organization is a requirement for the development of complex organisms, and filamentous cyanobacteria such as Anabaena represent a paradigmatic case of bacterial multicellularity. The Anabaena filament can include hundreds of communicated cells that exchange nutrients and regulators and, depending on environmental conditions, can include different cell types specialized in distinct biological functions. Hence, the specific features of the Anabaena filament and how they are propagated during cell division represent outstanding biological issues. Here, we studied SepT, a novel coiled-coil-rich protein of Anabaena that is located in the intercellular septa and influences the formation of the septal specialized structures that allow communication between neighboring cells along the filament, a fundamental trait for the performance of Anabaena as a multicellular organism.

The Role of MreB, MreC and MreD in the Morphology of the Diazotrophic Filament of Anabaena sp. PCC 7120.

The cyanobacterium Anabaena sp. PCC 7120 forms filaments of communicating cells. Under conditions of nitrogen scarcity, some cells differentiate into heterocysts, allowing the oxygen-sensitive N2-reduction system to be expressed and operated in oxic environments. The key to diazotrophic growth is the exchange of molecules with nutritional and signaling functions between the two types of cells of the filament. During heterocyst differentiation, the peptidoglycan sacculus grows to allow cell enlargement, and the intercellular septa are rebuilt to narrow the contact surface with neighboring cells and to hold specific transport systems, including the septal junction complexes for intercellular molecular transfer, which traverse the periplasm between heterocysts and neighboring vegetative cells through peptidoglycan nanopores. Here we have followed the spatiotemporal pattern of peptidoglycan incorporation during heterocyst differentiation by Van-FL labeling and the localization and role of proteins MreB, MreC and MreD. We observed strong transitory incorporation of peptidoglycan in the periphery and septa of proheterocysts and a maintained focal activity in the center of mature septa. During differentiation, MreB, MreC and MreD localized throughout the cell periphery and at the cell poles. In mreB, mreC or mreD mutants, instances of strongly increased peripheral and septal peptidoglycan incorporation were detected, as were also heterocysts with aberrant polar morphology, even producing filament breakage, frequently lacking the septal protein SepJ. These results suggest a role of Mre proteins in the regulation of peptidoglycan growth and the formation of the heterocyst neck during differentiation, as well as in the maintenance of polar structures for intercellular communication in the mature heterocyst. Finally, as previously observed in filaments growing with combined nitrogen, in the vegetative cells of diazotrophic filaments, the lack of MreB, MreC or MreD led to altered localization of septal peptidoglycan-growth bands reproducing an altered localization of FtsZ and ZipN rings during cell division.

The Role of Mre Factors and Cell Division in Peptidoglycan Growth in the Multicellular Cyanobacterium Anabaena.

Bacteria in general serve two main tasks: cell growth and division. Both processes include peptidoglycan extension to allow cell expansion and to form the poles of the daughter cells, respectively. The cyanobacterium Anabaena forms filaments of communicated cells in which the outer membrane and the peptidoglycan sacculus, which is engrossed in the intercellular regions between contiguous cells, are continuous along the filament. During the growth of Anabaena, peptidoglycan incorporation was weak at the cell periphery. During cell division, midcell peptidoglycan incorporation matched the localization of the divisome, and incorporation persisted in the intercellular septa, even after the division was completed. MreB, MreC, and MreD were located throughout the cell periphery and, in contrast to other bacteria, also to the divisome all along midcell peptidoglycan growth. In Anabaena mutants bearing inactivated mreB, mreC, or mreD genes, which showed conspicuous alterations in the filament morphology, consecutive septal bands of peptidoglycan growth were frequently not parallel to each other and were irregularly spaced along the filament, reproducing the disposition of the Z-ring. Both lateral and septal growth was impaired in strains down-expressing Z-ring components, and MreB and MreD appeared to directly interact with some divisome components. We propose that, in Anabaena, association with the divisome is a way for localization of MreB, MreC, and MreD at the cell poles, where they regulate lateral, midcell, and septal peptidoglycan growth with the latter being involved in localization and maintenance of the intercellular septal-junction protein structures that mediate cell-cell communication along the filament. IMPORTANCE Peptidoglycan surrounds the bacterial cell, being essential for the determination of the bacterium-specific morphology and survival. Peptidoglycan growth has been thoroughly investigated in some model rod-shaped bacteria, and more recently some representatives with disparate morphologies became into focus, revealing that patterns of peptidoglycan growth are much more diverse than previously anticipated. Anabaena forms filaments of communicated cells exhibiting features of multicellular organisms, such as the production of morphogens and coupled circadian oscillations. Here, we showed that Anabaena presented a distinct pattern of peptidoglycan growth characterized by continuous incorporation of material at the polar intercellular regions, contributing to assembling and maintaining the protein complexes that expand the septal peptidoglycan mediating intercellular molecular exchange in the filament.

Antimicrobial Activity of the Circular Bacteriocin AS-48 against Clinical Multidrug-Resistant Staphylococcus aureus.

The treatment and hospital-spread-control of methicillin-resistant Staphylococcus aureus (MRSA) is an important challenge since these bacteria are involved in a considerable number of nosocomial infections that are difficult to treat and produce prolonged hospitalization, thus also increasing the risk of death. In fact, MRSA strains are frequently resistant to all ß-lactam antibiotics, and co-resistances with other drugs such as macrolides, aminoglycosides, and lincosamides are usually reported, limiting the therapeutical options. To this must be added that the ability of these bacteria to form biofilms on hospital surfaces and devices confer high antibiotic resistance and favors horizontal gene transfer of genetic-resistant mobile elements, the spreading of infections, and relapses. Here, we genotypically and phenotypically characterized 100 clinically isolated S. aureus for their resistance to 18 antibiotics (33% of them were OXA resistant MRSA) and ability to form biofilms. From them, we selected 48 strains on the basis on genotype group, antimicrobial-resistance profile, and existing OXA resistance to be assayed against bacteriocin AS-48. The results showed that AS-48 was active against all strains, regardless of their clinical source, genotype, antimicrobial resistance profile, or biofilm formation capacity, and this activity was enhanced in the presence of the antimicrobial peptide lysozyme. Finally, we explored the effect of AS-48 on formed S. aureus biofilms, observing a reduction in S. aureus S-33 viability. Changes in the matrix structure of the biofilms as well as in the cell division process were observed with scanning electron microscopy in both S-33 and S-48 S. aureus strains.

The Inorganic Nutrient Regime and the mre Genes Regulate Cell and Filament Size and Morphology in the Phototrophic Multicellular Bacterium Anabaena.

The model cyanobacterium Anabaena sp. PCC 7120 exhibits a phototrophic metabolism relying on oxygenic photosynthesis and a complex morphology. The organismic unit is a filament of communicated cells that may include cells specialized in different nutritional tasks, thus representing a paradigm of multicellular bacteria. In Anabaena, the inorganic carbon and nitrogen regime influenced not only growth, but also cell size, cell shape, and filament length, which also varied through the growth cycle. When using combined nitrogen, especially with abundant carbon, cells enlarged and elongated during active growth. When fixing N2, which imposed lower growth rates, shorter and smaller cells were maintained. In Anabaena, gene homologs to mreB, mreC, and mreD form an operon that was expressed at higher levels during the phase of fastest growth. In an ntcA mutant, mre transcript levels were higher than in the wild type and, consistently, cells were longer. Negative regulation by NtcA can explain that Anabaena cells were longer in the presence of combined nitrogen than in diazotrophic cultures, in which the levels of NtcA are higher. mreB, mreC, and mreD mutants could grow with combined nitrogen, but only the latter mutant could grow diazotrophically. Cells were always larger and shorter than wild-type cells, and their orientation in the filament was inverted. Consistent with increased peptidoglycan width and incorporation in the intercellular septa, filaments were longer in the mutants, suggesting a role for MreB, MreC, and MreD in the construction of septal peptidoglycan that could affect intercellular communication required for diazotrophic growth.IMPORTANCE Most studies on the determination of bacterial cell morphology have been conducted in heterotrophic organisms. Here, we present a study of how the availability of inorganic nitrogen and carbon sources influence cell size and morphology in the context of a phototrophic metabolism, as found in the multicellular cyanobacterium Anabaena In Anabaena, the expression of the MreB, MreC, and MreD proteins, which influence cell size and length, are regulated by NtcA, a transcription factor that globally coordinates cellular responses to the C-to-N balance of the cells. Moreover, MreB, MreC, and MreD also influence septal peptidoglycan construction, thus affecting filament length and, possibly, intercellular molecular exchange that is required for diazotrophic growth. Thus, here we identified new roles for Mre proteins in relation to the phototrophic and multicellular character of a cyanobacterium, Anabaena.

Interactions of PatA with the Divisome during Heterocyst Differentiation in Anabaena.

The Anabaena organismic unit is a filament of communicating cells. Under conditions of nitrogen scarcity, some cells along the filament differentiate into heterocysts, which are specialized in the fixation of atmospheric N2 and provide the vegetative cells with N2 fixation products. At a certain stage, the differentiation process becomes irreversible, so that even when nitrogen is replenished, no return to the vegetative cell state takes place, possibly as a consequence of loss of cell division capacity. Upon N-stepdown, midcell FtsZ-rings were detected in vegetative cells, but not in differentiating cells, and this was also the case for ZipN, an essential protein that participates in FtsZ tethering to the cytoplasmic membrane and divisome organization. Later, expression of ftsZ was arrested in mature heterocysts. PatA is a protein required for the differentiation of intercalary heterocysts in Anabaena The expression level of the patA gene was increased in differentiating cells, and a mutant strain lacking PatA exhibited enhanced FtsZ-rings. PatA was capable of direct interactions with ZipN and SepF, another essential component of the Anabaena Z-ring. Thus, PatA appears to promote inhibition of cell division in the differentiating cells, allowing progress of the differentiation process. PatA, which in mature heterocysts was detected at the cell poles, could interact also with SepJ, a protein involved in production of the septal junctions that provide cell-cell adhesion and intercellular communication in the filament, hinting at a further role of PatA in the formation or stability of the intercellular structures that are at the basis of the multicellular character of AnabaenaIMPORTANCEAnabaena is a cyanobacterial model that represents an ancient and simple form of biological multicellularity. The Anabaena organism is a filament of cohesive and communicating cells that can include cells specialized in different tasks. Thus, under conditions of nitrogen scarcity, certain cells of the filament differentiate into heterocysts, which fix atmospheric nitrogen and provide organic nitrogen to the rest of cells, which, in turn, provide heterocysts with organic carbon. Heterocyst differentiation involves extensive morphological, biochemical, and genetic changes, becoming irreversible at a certain stage. We studied the regulation during heterocyst differentiation of several essential components of the Anabaena cell division machinery and found that protein PatA, which is required for differentiation and is induced in differentiating cells, interacts with essential cell division factors and destabilizes the cell division complex. This suggests a mechanism for establishment of commitment to differentiation by inhibition of cell division.