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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ABSTRACT: Within the past 10 years, it has become increasingly evident that posttranscriptional regulation is among the most important mechanisms used by bacteria to modulate gene expression in response to environmental perturbations. Posttranscriptional mechanisms provide a much faster response and lower energy burden compared with most transcription regulatory pathways, and they have the unique advantage that they can override existing transcriptional responses once the environment changes. Because of this, virulence factor gene expression is particularly suited for posttranscriptional control, and not surprisingly, an abundance of recent evidence indicates that posttranscriptional regulators are the predominant virulence regulators of human pathogens. Typically, this involves global riboregulators that primarily serve as modulators of virulence gene translation initiation and/or mRNA stability. Surprisingly little has been reported about posttranscriptional regulatory pathways in oral bacteria, but recent results suggest that oral species are equally dependent upon posttranscriptional control of their adaptive genetic responses. In this report, we discuss the major themes in RNA-based regulation of gene expression and review the available literature related to the most commonly studied oral bacterial species.Current Oral Health Reports. 03/2014; 1(1).
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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. · 3.53 Impact Factor
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ABSTRACT: Periodontitis is a common inflammatory disease affecting the tooth-supporting structures. It is initiated by bacteria growing as a biofilm at the gingival margin, and communication of the biofilms differs in health and disease. The bacterial composition of periodontitis-associated biofilms has been well documented and is under continual investigation. However, the roles of several host response and inflammation driven environmental stimuli on biofilm formation is not well understood. This review article addresses the effects of environmental factors such as pH, temperature, cytokines, hormones, and oxidative stress on periodontal biofilm formation and bacterial virulence.International Journal of Molecular Sciences 08/2013; 14(8):17221-37. · 2.34 Impact Factor