Salmonella Biofilm Formation under Fluidic Shear Stress on Different Surface Materials.

This study characterized biofilm formation of various Salmonella strains on common processing plant surface materials (stainless steel, concrete, rubber, polyethylene) under static and fluidic shear stress conditions. Surface-coupons were immersed in well-plates containing 1 mL of Salmonella (6 log CFU/mL) and incubated aerobically for 48 h at 37 °C in static or shear stress conditions. Biofilm density was determined using crystal violet assay, and biofilm cells were enumerated by plating on tryptic soy agar plates. Biofilms were visualized using scanning electron microscopy. Data were analyzed by SAS 9.4 at a significance level of 0.05. A surface-incubation condition interaction was observed for biofilm density (p < 0.001). On stainless steel, the OD600 was higher under shear stress than static incubation; whereas, on polyethylene, the OD600 was higher under static condition. Enumeration revealed surface-incubation condition (p = 0.024) and surface-strain (p < 0.001) interactions. Among all surface-incubation condition combinations, the biofilm cells were highest on polyethylene under fluidic shear stress (6.4 log/coupon; p < 0.001). Biofilms of S. Kentucky on polyethylene had the highest number of cells (7.80 log/coupon) compared to all other strain-surface combinations (p < 0.001). Electron microscopy revealed morphological and extracellular matrix differences between surfaces. Results indicate that Salmonella biofilm formation is influenced by serotype, surface, and fluidic shear stress.

Streptomyces spiramenti sp. nov., isolated from a deep-sea microbial mat.

Strain 5675061T was isolated from a deep-sea microbial mat near hydrothermal vents within the Axial Seamount caldera on the Juan de Fuca Ridge (NE Pacific Ocean) and was taxonomically evaluated using a polyphasic approach. Morphological and chemotaxonomic properties are consistent with characteristics of the genus Streptomyces: aerobic Gram-stain-positive filaments that form spores, L,L-diaminopimelic acid in whole-cell hydrolysates, and iso-C16:0 as the major fatty acid. Phylogenetic analysis, genomic, and biochemical comparisons show close evolutionary relatedness to Streptomyces lonarensis NCL716T, S. bohaiensis 11A07T, and S. otsuchiensis OTB305T but genomic relatedness indices identify strain 5675061T as a distinct species. Based on a polyphasic characterization, identifying differences in genomic and taxonomic data, strain 5675061T represents a novel species, for which the name Streptomyces spiramenti sp. nov. is proposed. The type strain is 5675061T (=LMG 31896T = DSM 111793T).

Intrafollicular injection of nanomolecules for advancing knowledge on folliculogenesis in livestock.

Despite the progress in assisted reproductive techniques, there is still a lack of rapid and minimally invasive in situ approaches for further enhancements of female fertility. Therefore, we synthesized clinically relevant liposome nanoparticles for ovarian intrafollicular injection to allow in vivo cellular imaging for future drug delivery, using the mare as an animal model. Ovarian follicles of living mares were injected in vivo with fluorescently labeled liposomes. Samples of the follicular wall (mural granulosa, theca interna, and theca externa), granulosa cells, and follicular fluid were harvested 24 h post-injection through the follicle wall biopsy (FWB), flushing, and aspiration techniques, respectively, using a transvaginal ultrasound-guided approach. In parallel, post-mortem dissected, and cultured porcine antral follicles were microinjected with doxorubicin-encapsulated liposomes to assess intracellular delivery potential. All injected mare and pig follicles were macroscopically healthy, and fluorescence imaging revealed successful intrafollicular binding to mural granulosa cells and progressive migration of liposomes to other follicle cell layers (theca interna, and theca externa), regardless of the follicle size. Intracellular delivery of doxorubicin was confirmed in all porcine follicle wall cell types. We conclude that the intrafollicular injection of nanomolecules is a promising approach for real-time monitoring of intrafollicular processes and potential utilization of in vivo cellular drug delivery to assist in follicle disease treatments and fertility improvement.

Mineralizing Pulmonary Elastosis in a Cat.

Mineralizing pulmonary elastosis (MPE) is a rare and unique phenomenon that has been reported in humans, typically secondary to recurrent pulmonary haemorrhage. MPE has a complex histopathological appearance, often containing iron-calcium deposits that can be mistaken as fungal organisms or other inorganic material. This report documents the first case of MPE in an animal species. A 10-year-old female domestic cat with respiratory failure was submitted for necropsy. The lungs were consolidated with severe pulmonary haemosiderosis, and widely disseminated granulomas surrounded large aggregates of hyphae-like structures. The pulmonary vasculature and airway smooth muscle were partially mineralized and fragmented. Histochemical stains revealed that the fungus-like material stained strongly with Prussian blue and alizarin red but only sparingly with von Kossa and negative with Gomori's methenamine silver stain. These findings are similar to those of MPE in humans. As most veterinary pathologists may not be familiar with MPE, it is important to avoid possible misinterpretation by recognizing its distinct features and the ancillary testing that may be required.

Phenotypic Assessment Suggests Multiple Start Codons for HetN, an Inhibitor of Heterocyst Differentiation, in Anabaena sp. Strain PCC 7120.

Multicellular organisms must carefully regulate the timing, number, and location of specialized cellular development. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen-fixing heterocysts are interspersed between vegetative cells in a periodic pattern to achieve an optimal exchange of bioavailable nitrogen and reduced carbon. The spacing between heterocysts is regulated by the activity of two developmental inhibitors, PatS and HetN. PatS functions to create a de novo pattern from a homogenous field of undifferentiated cells, while HetN maintains the pattern throughout subsequent growth. Both PatS and HetN harbor the peptide motif ERGSGR, which is sufficient to inhibit development. While the small size of PatS makes the interpretation of inhibitory domains relatively simple, HetN is a 287-amino-acid protein with multiple functional regions. Previous work suggested the possibility of a truncated form of HetN containing the ERGSGR motif as the source of the HetN-derived inhibitory signal. In this work, we present evidence that the glutamate of the ERGSGR motif is required for proper HetN inhibition of heterocysts. Mutational analysis and subcellular localization indicate that the gene encoding HetN uses two methionine start codons (M1 and M119) to encode two protein forms: M1 is required for protein localization, while M119 is primarily responsible for inhibitory function. Finally, we demonstrate that patS and hetN are not functionally equivalent when expressed from the other gene's regulatory sequences. Taken together, these results help clarify the functional forms of HetN and will help refine future work defining a HetN-derived inhibitory signal in this model of one-dimensional periodic patterning.IMPORTANCE The proper placement of different cell types during a developmental program requires the creation and maintenance of a biological pattern to define the cells that will differentiate. Here we show that the HetN inhibitor, responsible for pattern maintenance of specialized nitrogen-fixing heterocyst cells in the filamentous cyanobacterium Anabaena, may be produced from two different start methionine codons. This work demonstrates that the two start sites are individually involved in a different HetN function, either membrane localization or inhibition of cellular differentiation.

The hetZ gene indirectly regulates heterocyst development at the level of pattern formation in Anabaena sp. strain PCC 7120.

Multicellular development requires the careful orchestration of gene expression to correctly create and position specialized cells. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen-fixing heterocysts are differentiated from vegetative cells in a reproducibly periodic and physiologically relevant pattern. While many genetic factors required for heterocyst development have been identified, the role of HetZ has remained unclear. Here, we present evidence to clarify the requirement of hetZ for heterocyst production and support a model where HetZ functions in the patterning stage of differentiation. We show that a clean, nonpolar deletion of hetZ fails to express the developmental genes hetR, patS, hetP and hetZ correctly and fails to produce heterocysts. Complementation and overexpression of hetZ in a hetP mutant revealed that hetZ was incapable of bypassing hetP, suggesting that it acts upstream of hetP. Complementation and overexpression of hetZ in a hetR mutant, however, demonstrated bypass of hetR, suggesting that it acts downstream of hetR and is capable of bypassing the need for hetR for differentiation irrespective of nitrogen status. Finally, protein-protein interactions were observed between HetZ and HetR, Alr2902 and HetZ itself. Collectively, this work suggests a regulatory role for HetZ in the patterning phase of cellular differentiation in Anabaena.

Pseudoalteromonas piratica strain OCN003 is a coral pathogen that causes a switch from chronic to acute Montipora white syndrome in Montipora capitata.

Reports of mass coral mortality from disease have increased over the last two decades. Montipora white syndrome (MWS) is a tissue loss disease that has negatively impacted populations of the coral Montipora capitata in Kane'ohe Bay, Hawai'i. Two types of MWS have been documented; a progressive disease termed chronic MWS (cMWS), that can be caused by Vibrio owensii strain OCN002, and a comparatively faster disease termed acute MWS (aMWS), that can be caused by Vibrio coralliilyticus strain OCN008. M. capitata colonies exhibiting cMWS can spontaneously switch to aMWS in the field. In this study, a novel Pseudoalteromonas species, P. piratica strain OCN003, fulfilled Koch's postulates of disease causation as another etiological agent of aMWS. Additionally, OCN003 induced a switch from cMWS to aMWS on M. capitata in laboratory infection trials. A comparison of OCN003 and Vibrio coralliilyticus strain OCN008, showed that OCN003 was more effective at inducing the cMWS to aMWS switch in M. capitata than OCN008. This study is the first to demonstrate that similar disease signs on one coral species (aMWS on M. capitata) can be caused by multiple pathogens, and describes the first Pseudoalteromonas species that infects coral.

The heterocyst regulatory protein HetP and its homologs modulate heterocyst commitment in Anabaena sp. strain PCC 7120.

The commitment of differentiating cells to a specialized fate is fundamental to the correct assembly of tissues within a multicellular organism. Because commitment is often irreversible, entry into and progression through this phase of development must be tightly regulated. Under nitrogen-limiting conditions, the multicellular cyanobacterium Anabaena sp. strain PCC 7120 terminally commits ∼10% of its cells to become specialized nitrogen-fixing heterocysts. Although commitment is known to occur 9-14 h after the induction of differentiation, the factors that regulate the initiation and duration of this phase have yet to be elucidated. Here, we report the identification of four genes that share a functional domain and modulate heterocyst commitment: hetP (alr2818), asl1930, alr2902, and alr3234 Epistatic relationships between all four genes relating to commitment were revealed by deleting them individually and in combination; asl1930 and alr3234 acted most upstream to delay commitment, alr2902 acted next in the pathway to inhibit development, and hetP acted most downstream to drive commitment forward. Possible protein-protein interactions between HetP, its homologs, and the heterocyst master regulator, HetR, were assessed, and interaction partners were defined. Finally, patterns of gene expression for each homolog, as determined by promoter fusions to gfp and reverse transcription-quantitative PCR, were distinct from that of hetP in both spatiotemporal organization and regulation. We posit that a dynamic succession of protein-protein interactions modulates the timing and efficiency of the commitment phase of development and note that this work highlights the utility of a multicellular cyanobacterium as a model for the study of developmental processes.

Mutation of the murC and murB Genes Impairs Heterocyst Differentiation in Anabaena sp. Strain PCC 7120.

UNLABELLED: To stabilize cellular integrity in the face of environmental perturbations, most bacteria, including cyanobacteria, synthesize and maintain a strong, flexible, three-dimensional peptidoglycan lattice. Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium capable of differentiating morphologically distinct nitrogen-fixing heterocyst cells in a periodic pattern. While heterocyst development has been shown to require proper peptidoglycan remodeling, the role of peptidoglycan synthesis has remained unclear. Here we report the identification of two peptidoglycan synthesis genes, murC (alr5065) and murB (alr5066), as required for heterocyst development. The murC and murB genes are predicted to encode a UDP-N-acetylmuramate:L-alanine ligase and a UDP-N-acetylenolpyruvoylglucosamine reductase, respectively, and we confirm enzymatic function through complementation of Escherichia coli strains deficient for these enzymes. Cells depleted of either murC or murB expression failed to differentiate heterocysts under normally inducing conditions and displayed decreased filament integrity. To identify the stage(s) of development affected by murC or murB depletion, the spatial distribution of expression of the patterning marker gene, patS, was examined. Whereas murB depletion did not affect the pattern of patS expression, murC depletion led to aberrant expression of patS in all cells of the filament. Finally, expression of gfp controlled by the region of DNA immediately upstream of murC was enriched in differentiating cells and was repressed by the transcription factor NtcA. Collectively, the data in this work provide evidence for a direct link between peptidoglycan synthesis and the maintenance of a biological pattern in a multicellular organism. IMPORTANCE: Multicellular organisms that differentiate specialized cells must regulate morphological changes such that both cellular integrity and the dissemination of developmental signals are preserved. Here we show that the multicellular bacterium Anabaena, which differentiates a periodic pattern of specialized heterocyst cells, requires peptidoglycan synthesis by the murine ligase genes murC (alr5065) and murB (alr5066) for maintenance of patterned gene expression, filament integrity, and overall development. This work highlights the significant influence that intracellular structure and intercellular connections can have on the execution of a developmental program.

ABC Transporter Required for Intercellular Transfer of Developmental Signals in a Heterocystous Cyanobacterium.

UNLABELLED: In the filamentous cyanobacterium Anabaena, patS and hetN encode peptide-derived signals with many of the properties of morphogens. These signals regulate the formation of a periodic pattern of heterocysts by lateral inhibition of differentiation. Here we show that intercellular transfer of the patS- and hetN-dependent developmental signals from heterocysts to vegetative cells requires HetC, a predicted ATP-binding cassette transporter (ABC transporter). Relative to the wild type, in a hetC mutant differentiation resulted in a reduced number of heterocysts that were incapable of nitrogen fixation, but deletion of patS or hetN restored heterocyst number and function in a hetC background. These epistasis results suggest that HetC is necessary for conferring self-immunity to the inhibitors on differentiating cells. Nine hours after induction of differentiation, HetC was required for neither induction of transcription of patS nor intercellular transfer of the patS-encoded signal to neighboring cells. Conversely, in strains lacking HetC, the patS- and hetN-encoded signals were not transferred from heterocyst cells to adjacent vegetative cells. The results support a model in which the patS-dependent signal is initially transferred between vegetative cells in a HetC-independent fashion, but some time before morphological differentiation of heterocysts is complete, transfer of both signals transitions to a HetC-dependent process. IMPORTANCE: How chemical cues that regulate pattern formation in multicellular organisms move from one cell to another is a central question in developmental biology. In this study, we show that an ABC transporter, HetC, is necessary for transport of two developmental signals between different types of cells in a filamentous cyanobacterium. ABC transporters are found in organisms as diverse as bacteria and humans and, as the name implies, are often involved in the transport of molecules across a cellular membrane. The activity of HetC was shown to be required for signaling between heterocysts, which supply fixed nitrogen to the organism, and other cells, as well as for conferring immunity to self-signaling on developing heterocysts.

The trpE gene negatively regulates differentiation of heterocysts at the level of induction in Anabaena sp. strain PCC 7120.

Levels of 2-oxoglutarate (2-OG) reflect nitrogen status in many bacteria. In heterocystous cyanobacteria, a spike in the 2-OG level occurs shortly after the removal of combined nitrogen from cultures and is an integral part of the induction of heterocyst differentiation. In this work, deletion of one of the two annotated trpE genes in Anabaena sp. strain PCC 7120 resulted in a spike in the 2-OG level and subsequent differentiation of a wild-type pattern of heterocysts when filaments of the mutant were transferred from growth on ammonia to growth on nitrate. In contrast, 2-OG levels were unaffected in the wild type, which did not differentiate under the same conditions. An inverted-repeat sequence located upstream of trpE bound a central regulator of differentiation, HetR, in vitro and was necessary for HetR-dependent transcription of a reporter fusion and complementation of the mutant phenotype in vivo. Functional complementation of the mutant phenotype with the addition of tryptophan suggested that levels of tryptophan, rather than the demonstrated anthranilate synthase activity of TrpE, mediated the developmental response of the wild type to nitrate. A model is presented for the observed increase in 2-OG in the trpE mutant.

Mutation of sepJ reduces the intercellular signal range of a hetN-dependent paracrine signal, but not of a patS-dependent signal, in the filamentous cyanobacterium Anabaena sp. strain PCC 7120.

Formation and maintenance of a periodic pattern of nitrogen-fixing cells called heterocysts by the filamentous cyanobacterium Anabaena sp. strain PCC 7120 is dependent on regulators encoded by patS and hetN. In this study, genetic mosaic filaments that consisted of cells engineered to produce one of the developmental regulators flanked by target cells capable of reporting the activity of the developmental regulator were used to investigate the intercellular movement of patS- and hetN-dependent activity. We provide evidence that hetN encodes a paracrine signal with a signal range of several cells. The signal that moved between cells did not include the C-terminus of the annotated HetN protein as indicated by similar signal ranges from source cells expressing either hetN-YFP or hetN alone, despite a lack of intercellular exchange of the HetN-YFP fusion protein. Deletion of sepJ, which has been shown to encode a component of intercellular channels, caused a significant decrease in the signal range of hetN expressed from source cells but not of patS. These results are consistent with symplastic transport of a paracrine hetN-dependent signal between vegetative cells of Anabaena.

Expanding the direct HetR regulon in Anabaena sp. strain PCC 7120.

In response to a lack of environmental combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 differentiates nitrogen-fixing heterocyst cells in a periodic pattern. HetR is a transcription factor that coordinates the regulation of this developmental program. An inverted repeat-containing sequence in the hepA promoter required for proheterocyst-specific transcription was identified based on sequence similarity to a previously characterized binding site for HetR in the promoter of hetP. The binding affinity of HetR for the hepA site is roughly an order of magnitude lower than that for the hetP binding site. A BLAST search of the Anabaena genome identified 166 hepA-like sites that occur as single or tandem sites (two binding sites separated by 13 bp). The vast majority of these sites are present in predicted intergenic regions. HetR bound five representative single binding sites in vitro, and binding was abrogated by transversions in the binding sites that conserved the inverted repeat nature of the sites. Binding to four representative tandem sites was not observed. Transcriptional fusions of the green fluorescent protein gene gfp with putative promoter regions associated with the representative binding sites indicated that HetR could function as either an activator or repressor and that activation was cell-type specific. Taken together, we have expanded the direct HetR regulon and propose a model in which three categories of HetR binding sites, based on binding affinity and nucleotide sequence, contribute to three of the four phases of differentiation.

The RGSGR amino acid motif of the intercellular signalling protein, HetN, is required for patterning of heterocysts in Anabaena sp. strain PCC 7120.

Nitrogen-fixing heterocysts are arranged in a periodic pattern on filaments of the cyanobacterium Anabaena sp. strain PCC 7120 under conditions of limiting combined nitrogen. Patterning requires two inhibitors of heterocyst differentiation, PatS and HetN, which work at different stages of differentiation by laterally suppressing levels of an activator of differentiation, HetR, in cells adjacent to source cells. Here we show that the RGSGR sequence in the 287-amino-acid HetN protein, which is shared by PatS, is critical for patterning. Conservative substitutions in any of the five amino acids lowered the extent to which HetN inhibited differentiation when overproduced and altered the pattern of heterocysts in filaments with an otherwise wild-type genetic background. Conversely, substitution of amino acids comprising the putative catalytic triad of this predicted reductase had no effect on inhibition or patterning. Deletion of putative domains of HetN suggested that the RGSGR motif is the primary component of HetN required for both its inhibitory and patterning activity, and that localization to the cell envelope is not required for patterning of heterocysts. The intercellular signalling proteins PatS and HetN use the same amino acid motif to regulate different stages of heterocyst patterning.