title: RNA-based molecular survey of biodiversity of limestone tombstone microbiota in response to atmospheric sulphur pollution.
abstract: Outdoor stoneworks sustain biofilm formation and are constantly at risk of deterioration by micro-organisms. In this study, the biofilm microflora of historic limestone tombstones located in a highly polluted urban environment (Cambridge, MA) and in a less polluted location (Lexington, MA) were compared using comprehensive RNA-based molecular analyses of 16S rRNA gene sequences as well as sequences of genes for different pathways of sulphur metabolism (soxB, apsA, dsrA). The metabolically active micro-organisms detected by denaturing gradient gel electrophoresis analysis of 16S rRNA fragments were predominantly represented by cyanobacteria (belonging to the family Nostocaceae and to the genus Chroococcidiopsis) in both polluted and unpolluted environments. The investigation of soxB, apsA, dsrA transcripts reflected the abundance and the diversity of sulphur-oxidizing and sulphate-reducing bacteria in the Cambridge samples in comparison with the Lexington samples. The investigation revealed that in addition to phototrophic sulphur bacteria belonging to the genera Thiocapsa, Halochromatium, Allochromatium, Thiococcus and Thermochromatium, other sulphate-oxidizing prokaryotes (e.g. the genus Thiobacillus) as well as sequences of Deltaproteobacteria from the genus Desulfovibrio occurred at the polluted urban site. The interactions between the main functional groups retrieved from the limestone tombstones were discussed.
  
  
  CONCLUSIONS
  The biofilm microflora inhabiting historic limestones are a multi-component open ecosystem sensitively reacting to all environmental factors including air pollutants. Little is known about specific target groups that are active in the biofilm and their physiological functions. For the first time, transcripts involved in important energy-yielding processes were investigated to reveal the metabolic capabilities of the microflora in response to atmospheric sulphur pollution. This work provides novel and important information about the ecology of limestone tombstone microbiota and its complex interaction with the external environment.
authors.affiliation: Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente, Università degli Studi di Milano, Milano, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Villa
authors.forename: A
authors.initials: A
authors.lastname: Vasanthakumar
authors.forename: R
authors.initials: R
authors.lastname: Mitchell
authors.forename: F
authors.initials: F
authors.lastname: Cappitelli
mesh_terms: D000393:Air Pollutants / Q000378:metabolism*; D000397:Air Pollution; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002119:Calcium Carbonate; D020459:Genes, rRNA; D008404:Massachusetts; D064307:Microbiota; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013431:Sulfates / Q000378:metabolism; D013455:Sulfur / Q000378:metabolism*
chemical_list: D000393:Air Pollutants; D012336:RNA, Ribosomal, 16S; D013431:Sulfates; D013455:Sulfur; D002119:Calcium Carbonate
keywords: RNA-based molecular analysis; limestone tombstone; pollution; subaerial biofilm; sulphur metabolism