NosP Signaling Modulates the NO/H-NOX-Mediated Multicomponent c-Di-GMP Network and Biofilm Formation in Shewanella oneidensis.

Biofilms form when bacteria aggregate in a self-secreted exopolysaccharide matrix; they are resistant to antibiotics and implicated in disease. Nitric oxide (NO) is known to mediate biofilm formation in many bacteria via ligation to H-NOX (heme-NO/oxygen binding) domains. Most NO-responsive bacteria, however, lack H-NOX domain-containing proteins. We have identified another NO-sensing protein (NosP), which is predicted to be involved in two-component signaling and biofilm regulation in many species. Here, we demonstrate that NosP participates in the previously described H-NOX/NO-responsive multicomponent c-di-GMP signaling network in Shewanella oneidensis. Strains lacking either nosP or its co-cistronic kinase nahK (previously hnoS) produce immature biofilms, while hnoX and hnoK (kinase responsive to NO/H-NOX) mutants result in wild-type biofilm architecture. We demonstrate that NosP regulates the autophosphorylation activity of NahK as well as HnoK. HnoK and NahK have been shown to regulate three response regulators (HnoB, HnoC, and HnoD) that together comprise a NO-responsive multicomponent c-di-GMP signaling network. Here, we propose that NosP/NahK adds regulation on top of H-NOX/HnoK to modulate this c-di-GMP signaling network, and ultimately biofilm formation, by governing the flux of phosphate through both HnoK and NahK. In addition, it appears that NosP and H-NOX act to counter each other in a push-pull mechanism; NosP/NahK promotes biofilm formation through inhibition of H-NOX/HnoK signaling, which itself reduces the extent of biofilm formation. Addition of NO results in a reduction of c-di-GMP and biofilm formation, primarily through disinhibition of HnoK activity.

Characterization of ExeM, an Extracellular Nuclease of Shewanella oneidensis MR-1.

Bacterial extracellular nucleases have multiple functions in processes as diverse as nutrient acquisition, natural transformation, biofilm formation, or defense against neutrophil extracellular traps (NETs). Here we explored the properties of ExeM in Shewanella oneidensis MR-1, an extracellular nuclease, which is widely conserved among species of Shewanella, Vibrio, Aeromonas, and others. In S. oneidensis, ExeM is crucial for normal biofilm formation. In vitro activity measurements on heterologously produced ExeM revealed that this enzyme is a sugar-unspecific endonuclease, which requires Ca2+ and Mg2+/Mn2+ as co-factors for full activity. ExeM was almost exclusively localized to the cytoplasmic membrane fraction, even when a putative C-terminal membrane anchor was deleted. In contrast, ExeM was not detected in medium supernatants. Based on the results we hypothesize that ExeM predominantly interacts with DNA in close proximity to the cell, e.g., to promote biofilm formation and defense against NETs, or to control uptake of DNA.

Iron triggers λSo prophage induction and release of extracellular DNA in Shewanella oneidensis MR-1 biofilms.

Prophages are ubiquitous elements within bacterial chromosomes and affect host physiology and ecology in multiple ways. We have previously demonstrated that phage-induced lysis is required for extracellular DNA (eDNA) release and normal biofilm formation in Shewanella oneidensis MR-1. Here, we investigated the regulatory mechanisms of prophage λSo spatiotemporal induction in biofilms. To this end, we used a functional fluorescence fusion to monitor λSo activation in various mutant backgrounds and in response to different physiological conditions. λSo induction occurred mainly in a subpopulation of filamentous cells in a strictly RecA-dependent manner, implicating oxidative stress-induced DNA damage as the major trigger. Accordingly, mutants affected in the oxidative stress response (ΔoxyR) or iron homeostasis (Δfur) displayed drastically increased levels of phage induction and abnormal biofilm formation, while planktonic cells were not or only marginally affected. To further investigate the role of oxidative stress, we performed a mutant screen and identified two independent amino acid substitutions in OxyR (T104N and L197P) that suppress induction of λSo by hydrogen peroxide (H2O2). However, λSo induction was not suppressed in biofilms formed by both mutants, suggesting a minor role of intracellular H2O2 in this process. In contrast, addition of iron to biofilms strongly enhanced λSo induction and eDNA release, while both processes were significantly suppressed at low iron levels, strongly indicating that iron is the limiting factor. We conclude that uptake of iron during biofilm formation triggers λSo-mediated lysis of a subpopulation of cells, likely by an increase in iron-mediated DNA damage sensed by RecA.

Transcriptome analysis of early surface-associated growth of Shewanella oneidensis MR-1.

Bacterial biofilm formation starts with single cells attaching to a surface, however, little is known about the initial attachment steps and the adaptation to the surface-associated life style. Here, we describe a hydrodynamic system that allows easy harvest of cells at very early biofilm stages. Using the metal ion-reducing gammaproteobacterium Shewanella oneidensis MR-1 as a model organism, we analyzed the transcriptional changes occurring during surface-associated growth between 15 and 60 minutes after attachment. 230 genes were significantly upregulated and 333 were downregulated by a factor of ≥ 2. Main functional categories of the corresponding gene products comprise metabolism, uptake and transport, regulation, and hypothetical proteins. Among the genes highly upregulated those implicated in iron uptake are highly overrepresented, strongly indicating that S. oneidensis MR-1 has a high demand for iron during surface attachment and initial biofilm stages. Subsequent microscopic analysis of biofilm formation under hydrodynamic conditions revealed that addition of Fe(II) significantly stimulated biofilm formation of S. oneidensis MR-1 while planktonic growth was not affected. Our approach to harvest cells for transcriptional analysis of early biofilm stages is expected to be easily adapted to other bacterial species.

Functional specificity of extracellular nucleases of Shewanella oneidensis MR-1.

Bacterial species such as Shewanella oneidensis MR-1 require extracellular nucleolytic activity for the utilization of extracellular DNA (eDNA) as a source of nutrients and for the turnover of eDNA as a structural matrix component during biofilm formation. We have previously characterized two extracellular nucleases of S. oneidensis MR-1, ExeM and ExeS. Although both are involved in biofilm formation, they are not specifically required for the utilization of eDNA as a nutrient. Here we identified and characterized EndA, a third extracellular nuclease of Shewanella. The heterologously overproduced and purified protein was highly active and rapidly degraded linear and supercoiled DNAs of various origins. Divalent metal ions (Mg(2+) or Mn(2+)) were required for function. endA is cotranscribed with phoA, an extracellular phosphatase, and is not upregulated upon phosphostarvation. Deletion of endA abolished both extracellular degradation of DNA by S. oneidensis MR-1 and the ability to use eDNA as a sole source of phosphorus. PhoA is not strictly required for the exploitation of eDNA as a nutrient. The activity of EndA prevents the formation of large cell aggregates during planktonic growth. However, in contrast to the findings for ExeM, endA deletion had only minor effects on biofilm formation. The findings strongly suggest that the extracellular nucleases of S. oneidensis exert specific functions required under different conditions.

Analysis of the BarA/UvrY two-component system in Shewanella oneidensis MR-1.

The BarA/UvrY two-component system is well conserved in species of the γ-proteobacteria and regulates numerous processes predominantly by controlling the expression of a subset of noncoding small RNAs. In this study, we identified and characterized the BarA/UvrY two-component system in the gammaproteobacterium Shewanella oneidensis MR-1. Functional interaction of sensor kinase BarA and the cognate response regulator UvrY was indicated by in vitro phosphotransfer studies. The expression of two predicted small regulatory RNAs (sRNAs), CsrB1 and CsrB2, was dependent on UvrY. Transcriptomic analysis by microarrays revealed that UvrY is a global regulator and directly or indirectly affects transcript levels of more than 200 genes in S. oneidensis. Among these are genes encoding key enzymes of central carbon metabolism such as ackA, aceAB, and pflAB. As predicted of a signal transduction pathway that controls aspects of central metabolism, mutants lacking UvrY reach a significantly higher OD than the wild type during aerobic growth on N-acetylglucosamine (NAG) while under anaerobic conditions the mutant grew more slowly. A shorter lag phase occurred with lactate as carbon source. In contrast, significant growth phenotypes were absent in complex medium. Based on these studies we hypothesize that, in S. oneidensis MR-1, the global BarA/UvrY/Csr regulatory pathway is involved in central carbon metabolism processes.

ArcS, the cognate sensor kinase in an atypical Arc system of Shewanella oneidensis MR-1.

The availability of oxygen is a major environmental factor for many microbes, in particular for bacteria such as Shewanella species, which thrive in redox-stratified environments. One of the best-studied systems involved in mediating the response to changes in environmental oxygen levels is the Arc two-component system of Escherichia coli, consisting of the sensor kinase ArcB and the cognate response regulator ArcA. An ArcA ortholog was previously identified in Shewanella, and as in Escherichia coli, Shewanella ArcA is involved in regulating the response to shifts in oxygen levels. Here, we identified the hybrid sensor kinase SO_0577, now designated ArcS, as the previously elusive cognate sensor kinase of the Arc system in Shewanella oneidensis MR-1. Phenotypic mutant characterization, transcriptomic analysis, protein-protein interaction, and phosphotransfer studies revealed that the Shewanella Arc system consists of the sensor kinase ArcS, the single phosphotransfer domain protein HptA, and the response regulator ArcA. Phylogenetic analyses suggest that HptA might be a relict of ArcB. Conversely, ArcS is substantially different with respect to overall sequence homologies and domain organizations. Thus, we speculate that ArcS might have adopted the role of ArcB after a loss of the original sensor kinase, perhaps as a consequence of regulatory adaptation to a redox-stratified environment.