Novel iron-regulated and Fur-regulated small regulatory RNAs in Aggregatibacter actinomycetemcomitans
ABSTRACT Iron can regulate biofilm formation via non-coding small RNA (sRNA). To determine if iron-regulated sRNAs are involved in biofilm formation by the periodontopathogen Aggregatibacter actinomycetemcomitans, total RNA was isolated from bacteria cultured with iron supplementation or chelation. Transcriptional analysis demonstrated that the expression of four sRNA molecules (JA01-JA04) identified by bioinformatics was significantly upregulated in iron-limited medium compared with iron-rich medium. A DNA fragment encoding each sRNA promoter was able to titrate Escherichia coli ferric uptake regulator (Fur) from a Fur-repressible reporter fusion in an iron uptake regulator titration assay. Cell lysates containing recombinant AaFur shifted the mobility of sRNA-specific DNAs in a gel shift assay. Potential targets of these sRNAs, determined in silico, included genes involved in biofilm formation. The A. actinomycetemcomitans overexpressing JA03 sRNA maintained a rough phenotype on agar, but no longer adhered to uncoated polystyrene or glass, although biofilm determinant gene expression was only modestly decreased. In summary, these sRNAs have the ability to modulate biofilm formation, but their functional target genes remain to be confirmed.
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- "However there is a previous report of other iron-responsive sRNAs encoded by a Pasteurellaceae species. Work by Amarasinghe et al. reported the presence of four Fur-regulated sRNAs, JA01 through JA04, in Aggregatibacter actinomycetemcomitans (Aa) strain HK1651R . BLAST analysis of these four sRNAs reveled that JA01 is only conserved among Aa strains. "
ABSTRACT: Nontypeable Haemophilus influenzae (NTHi) are Gram-negative commensal bacteria that reside in the nasopharynx. NTHi can also cause multiple upper and lower respiratory tract diseases that include sinusitis, conjunctivitis, bronchitis, and otitis media. In numerous bacterial species the ferric uptake regulator (Fur) acts as a global regulator of iron homeostasis by negatively regulating the expression of iron uptake systems. However in NTHi strain 86-028NP and numerous other bacterial species there are multiple instances where Fur positively affects gene expression. It is known that many instances of positive regulation by Fur occur indirectly through a small RNA intermediate. However, no examples of small RNAs have been described in NTHi. Therefore we used RNA-Seq analysis to analyze the transcriptome of NTHi strain 86-028NPrpsL and an isogenic 86-028NPrpsLΔfur strain to identify Fur-regulated intergenic transcripts. From this analysis we identified HrrF, the first small RNA described in any Haemophilus species. Orthologues of this small RNA exist only among other Pasteurellaceae. Our analysis showed that HrrF is maximally expressed when iron levels are low. Additionally, Fur was shown to bind upstream of the hrrF promoter. RNA-Seq analysis was used to identify targets of HrrF which include genes whose products are involved in molybdate uptake, deoxyribonucleotide synthesis, and amino acid biosynthesis. The stability of HrrF is not dependent on the RNA chaperone Hfq. This study is the first step in an effort to investigate the role small RNAs play in altering gene expression in response to iron limitation in NTHi.PLoS ONE 08/2014; 9(8):e105644. DOI:10.1371/journal.pone.0105644 · 3.23 Impact Factor
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- "It is proposed that these ncRNAs could be interacting with biofilm-associated genes, including the flp fimbrial operon and genes associated with EPS, according to in silico models (Amarasinghe et al., 2012). So far the ability of these iron-regulated ncRNAs to modulate biofilm formation has been demonstrated, but their functional target genes remain to be elucidated (Amarasinghe et al., 2012). The ncRNAs are sometimes part of complex regulatory networks that finally induce biofilm formation by posttranscriptional control. "
ABSTRACT: Biofilms are characterized by a dense multicellular community of microorganisms that can be formed by the attachment of bacteria to an inert surface and to each other. The development of biofilm involves the initial attachment of planktonic bacteria to a surface, followed by replication, cell-to-cell adhesion to form microcolonies, maturation, and detachment. Mature biofilms are embedded in a self-produced extracellular polymeric matrix composed primarily of bacterial-derived exopolysaccharides, specialized proteins, adhesins, and occasionally DNA. Because the synthesis and assembly of biofilm matrix components is an exceptionally complex process, the transition between its different phases requires the coordinate expression and simultaneous regulation of many genes by complex genetic networks involving all levels of gene regulation. The finely controlled intracellular level of the chemical second messenger molecule, cyclic-di-GMP is central to the post-transcriptional mechanisms governing the switch between the motile planktonic lifestyle and the sessile biofilm forming state in many bacteria. Several other post-transcriptional regulatory mechanisms are known to dictate biofilm development and assembly and these include RNA-binding proteins, small non-coding RNAs, toxin-antitoxin systems, riboswitches, and RNases. Post-transcriptional regulation is therefore a powerful molecular mechanism employed by bacteria to rapidly adjust to the changing environment and to fine tune gene expression to the developmental needs of the cell. In this review, we discuss post-transcriptional mechanisms that influence the biofilm developmental cycle in a variety of pathogenic bacteria.Frontiers in Cellular and Infection Microbiology 03/2014; 4(4):38. DOI:10.3389/fcimb.2014.00038 · 3.72 Impact Factor
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ABSTRACT: Sal4 is a monoclonal, polymeric IgA antibody directed against the O-antigen (O-Ag) of Salmonella enterica serovar Typhimurium (S. Typhimurium) that is sufficient to protect mice against S. Typhimurium intestinal infection. We recently reported that exposure of S. Typhimurium to Sal4 results in the immediate loss of flagella-based motility, alterations in outer membrane (OM) integrity, and the concomitant appearance of a mucoid phenotype that is reminiscent of cells in the earliest stages of biofilm formation. We now demonstrate that prolonged (>4h) exposure of S. Typhimurium to Sal4 at 37°C (but not ambient temperature) results in measurable exopolysaccharide (EPS) accumulation and biofilm formation on borosilicate glass surfaces and polystyrene microtiter plates. The polysaccharide produced by S. Typhimurium in response to Sal4 contains cellulose, in addition to O-Ag capsule and colanic acid. EPS production was dependent on YeaJ, a proposed inner membrane-localized diguanylate cyclase (DGC) and a known regulator of cellulose biosynthesis. A ΔyeaJ strain was unable to produce cellulose or form a biofilm in response to Sal4. Conversely, over expression of yeaJ in S. Typhimurium enhanced Sal4-induced biofilm formation and resulted in increased intracellular levels of c-di-GMP, as compared to a wild-type control, strongly suggesting that YeaJ is indeed a functional DGC. Based on these data we speculate that Sal4, by virtue of its ability to associate with the O-Ag and induce OM stress, renders S. Typhimurium avirulent by triggering c-di-GMP-dependent signaling pathway via YeaJ that leads to suppression of bacterial motility, while simultaneously stimulating EPS production.Infection and immunity 12/2012; 81(3). DOI:10.1128/IAI.00813-12 · 3.73 Impact Factor