Shewanella oneidensis MR-1 is a facultative Fe(III)-and Mn(IV)-reducing microorganism and serves as a model for studying microbially induced dissolution of Fe or Mn oxide minerals as well as biogeochemical cycles. In soil and sediment environments, S. oneidensis biofilms form on mineral surfaces and are critical for mediating the metabolic interaction between this microbe and insoluble metal oxide phases. In order to develop an understanding of the molecular basis of biofilm formation, we investigated S. oneidensis biofilms developing on glass surfaces in a hydrodynamic flow chamber system. After initial attachment, growth of microcolonies and lateral spreading of biofilm cells on the surface occurred simultaneously within the first 24 h. Once surface coverage was almost complete, biofilm development proceeded with extensive vertical growth, resulting in formation of towering structures giving rise to pronounced three-dimensional architecture. Biofilm development was found to be dependent on the nutrient conditions, suggesting a metabolic control. In global transposon mutagenesis, 173 insertion mutants out of 15,000 mutants screened were identified carrying defects in initial attachment and/or early stages in biofilm formation. Seventy-one of those mutants exhibited a nonswimming phenotype, suggesting a role of swimming motility or motility elements in biofilm formation. Disruption mutations in motility genes (flhB, fliK, and pomA), however, did not affect initial attachment but affected progression of biofilm development into pronounced three-dimensional architecture. In contrast, mutants defective in mannose-sensitive hemagglutinin type IV pilus biosynthesis and in pilus retraction (pilT) showed severe defects in adhesion to abiotic surfaces and biofilm formation, respectively. The results provide a basis for understanding microbe-mineral interactions in natural environments.Microbe-mineral interactions are key steps in the biochemical cycles of elements. Of particular interest are microbemediated redox transformations of Fe and Mn oxide-containing minerals, which are abundantly present in soil as insoluble (oxyhydr)oxide minerals (e.g., goethite, ferrihydrite, and birnessite). In case of reductive interactions, microbially produced reactive Fe(II) and Mn(II) species dissolve from the mineral phase and can subsequently initiate geochemically important, abiotic reactions, such as the reductive transformation of Se, As, and Cr species (9,22,33). Shewanella oneidensis and Geobacter species are soil and sediment microbes that reduce insoluble Fe(III) and Mn(IV) in their catabolic metabolism under anaerobic conditions (24,34,35). Key to those microbemineral interactions, however, is the close association of the microbe with the mineral surface. In natural environments, such close contact is mediated by microbial biofilms, which develop on mineral particles and enable these biofilm microbes to metabolically interact with the metals of the mineral phase.Despite intense research over the past decade on medically impor...

Stability and resilience against environmental perturbations are critical properties of medical and environmental biofilms and pose important targets for their control. Biofilm stability is determined by two mutually exclusive processes: attachment of cells to and detachment from the biofilm matrix. Using Shewanella oneidensis MR-1, an environmentally versatile, Fe(III) and Mn(IV) mineral-reducing microorganism, we identified mxdABCD as a new set of genes essential for formation of a three-dimensional biofilm. Molecular analysis revealed that mxdA encodes a cyclic bis(3,5)guanylic acid (cyclic di-GMP)-forming enzyme with an unusual GGDEF motif, i.e., NVDEF, which is essential for its function. mxdB encodes a putative membrane-associated glycosyl transferase. Both genes are essential for matrix attachment. The attachment-deficient phenotype of a ⌬mxdA mutant was rescued by ectopic expression of VCA0956, encoding another diguanylate cyclase. Interestingly, a rapid cellular detachment from the biofilm occurred upon induction of yhjH, a gene encoding an enzyme that has been shown to have phosphodiesterase activity. In this way, it was possible to bypass the previously identified sudden depletion of molecular oxygen as an environmental trigger to induce biofilm dissolution. We propose a model for c-di-GMP as a key intracellular regulator for controlling biofilm stability by shifting the state of a biofilm cell between attachment and detachment in a concentration-dependent manner.

Active detachment of cells from microbial biofilms is a critical yet poorly understood step in biofilm development. We discovered that detachment of cells from biofilms of Shewanella oneidensis MR-1 can be induced by arresting the medium flow in a hydrodynamic biofilm system. Induction of detachment was rapid, and substantial biofilm dispersal started as soon as 5 min after the stop of flow. We developed a confocal laser scanning microscopy-based assay to quantify detachment. The extent of biomass loss was found to be dependent on the time interval of flow stop and on the thickness of the biofilm. Up to 80% of the biomass of 16-h-old biofilms could be induced to detach. High-resolution microscopy studies revealed that detachment was associated with an overall loosening of the biofilm structure and a release of individual cells or small cell clusters. Swimming motility was not required for detachment. Although the loosening of cells from the biofilm structure was observed evenly throughout thin biofilms, the most pronounced detachment in thicker biofilms occurred in regions exposed to the flow of medium, suggesting a metabolic control of detachability. Deconvolution of the factors associated with the stop of medium flow revealed that a sudden decrease in oxygen tension is the predominant trigger for initiating detachment of individual cells. In contrast, carbon limitation did not trigger any substantial detachment, suggesting a physiological link between oxygen sensing or metabolism and detachment. In-frame deletions were introduced into genes encoding the known and putative global transcriptional regulators ArcA, CRP, and EtrA (FNR), which respond to changes in oxygen tension in S. oneidensis MR-1. Biofilms of null mutants in arcA and crp were severely impacted in the stop-of-flow-induced detachment response, suggesting a role for these genes in regulation of detachment. In contrast, an ⌬etrA mutant displayed a variable detachment phenotype. From this genetic evidence we conclude that detachment is a biologically controlled process and that a rapid change in oxygen concentration is a critical factor in detachment and, consequently, in dispersal of S. oneidensis cells from biofilms. Similar mechanisms might also operate in other bacteria.

Stability and resistance to dissolution are key features of microbial biofilms. How these macroscopic properties are determined by the physiological state of individual biofilm cells in their local physical-chemical and cellular environment is largely unknown. In order to obtain molecular and energetic insight into biofilm stability, we investigated whether maintenance of biofilm stability is an energy-dependent process and whether transcription and/or translation is required for biofilm dissolution. We found that in 12-hour-old Shewanella oneidensis MR-1 biofilms, a reduction in cellular ATP concentration, induced either by oxygen deprivation or by addition of the inhibitor of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), dinitrophenol (DNP), or CN ؊ , resulted in massive dissolution. In 60-hour-old biofilms, the extent of uncouplerinduced cell loss was strongly attenuated, indicating that the integrity of older biofilms is maintained by means other than those operating in younger biofilms. In experiments with 12-hour-old biofilms, the transcriptional and translational inhibitors rifampin, tetracycline, and erythromycin were found to be ineffective in preventing energy starvation-induced detachment, suggesting that neither transcription nor translation is required for this process. Biofilms of Vibrio cholerae were also induced to dissolve upon CCCP addition to an extent similar to that in S. oneidensis. However, Pseudomonas aeruginosa and P. putida biofilms remained insensitive to CCCP addition. Collectively, our data show that metabolic energy is directly or indirectly required for maintaining cell attachment, and this may represent a common but not ubiquitous mechanism for stability of microbial biofilms.

Most of the phosphorus (P) in maize (Zea mays L.) kernels is in the form of phytic acid. A low phytic acid (lpa) maize mutant, lpa1-1, displays levels reduced by 66%. A goal of genetic breeding is development of low phytic acid feedstocks to improve P nutrition of nonruminant animals and reduce the adverse environmental impacts of excess P in manure. The genetic basis of the lpa1-1 mutation is not known, but previous genetic mapping has shown both the mutant phenotype and the Ins (3) P(1) synthase (MIPS) gene, which encodes the first enzyme, myo-inositol phosphate synthase, in the phytic acid biosynthetic pathway, map to the same chromosomal region in maize. Research was conducted to determine the expression of the MIPS gene in lpa1-1 and wild-type kernels with similar genetic backgrounds and to ascertain if variation in the MIPS coding sequence could be inferred to be the basis of the mutation. MIPS enzyme activity determined by TLC was reduced 2-3-fold in mutant kernels. RT-PCR-based experiments using primers specific for the 1S-MIPS sequence indicated gene expression was reduced 50-60% in the mutant. Sequence analysis of the MIPS genomic sequence revealed 10 exons and 9 introns that are identical in both mutant and wild-type developing kernels. These findings support an association between reduced MIPS gene activity and low phytic acid content, but additional research should examine the promoter, the 5'UTR, or transcriptional controlling elements of the MIPS gene to ascertain the possible presence of a genetic lesion in those regions.