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Cyanobacteria are prokaryotes capable of oxygenic photosynthesis, and for many species, nitrogen fixation, giving cyanobacteria an important role in global carbon and nitrogen cycles. Furthermore, multicellular filamentous cyanobacteria are developmentally complex, capable of differentiation into different cell types, including cells capable of nitrogen fixation and cells for motility, making them an ideal platform for studying development, as well as for practical use in biotechnology. Understanding how developmental programmes are activated require an understanding of the role of alternative sigma factors, which are required for transcriptional activation in bacteria. In order to investigate the gene regulatory network and to determine the role of alternative sigma factors in hormogonium development, real time PCR and Next Generation RNA-seq were used to measure expression levels of genes involved in hormogonium development and to further characterise the nature of the hormogonium developmental programme in the filamentous cyanobacterium Nostoc punctiforme. The results support a model where a hierarchal sigma factor cascade activates hormogonium development, in which expression of sigJ activates expression of the sigma factors sigC and sigF, as well as a wide range of other genes, including those involved in the type IV pilus (T4P), chemotaxis-like systems, and cell architecture. SigC and SigF have more limited roles: cell division genes are dependent on SigC and pilA expression was stringently SigF-dependent. Interestingly, SigC was also found to enhance expression of sigJ during hormogonium development, implying a potential positive feedback loop between sigJ and sigC.

The  type  III  secretion  system  (T3SS)  is  required  for  virulence  of  the  gram-­- negative   plant   pathogen   Pseudomonas  syringae   pv.   tomato  DC3000   (DC3000)   in   tomato   and   Arabidopsis.   The   alternative   sigma   factor   HrpL   directly   regulates   expression  of  T3SS  genes  by  binding  to  a  short  DNA  sequence  designated  as  the   "hrp  promoter".  The  ability  of  DC3000  to  colonize  plants,  subdue  multiple  layers   of  plant  defense  and  multiply  in  plant  tissues  relies  on  the  activities  carried  out   by  the  many  T3SS  regulon  members  (known  collectively  as  hrp  genes).     Efforts   to   identify   genes   involved   in   pathogenicity   were   initiated   over   three   decades   ago.   However,   HrpL   binding   to   hrp   promoters   has   never   been   directly   demonstrated   and   it   is   unclear   if   the   list   of   HrpL-­-regulated   genes   is   complete.   The   first   goal   of   the   research   described   here   was   to   systemically   and   exhaustively  identify  HrpL-­-binding  sites  and  likely  hrp  promoters  in  the  DC3000   genome.   Employing   chromatin   immuno-­-precipitation,   coupled   with   high-­- throughput   sequencing   (ChIP-­-Seq)   and   transcription   start   site   analysis   (modified   RNA-­-Seq),   we   found   twenty   sites   representing   novel   hrp   promoters.   Using   deletion   analysis,   we   attempted   to   determine   if   the   genes   downstream   from   a   subset   of   these   promoters   could   be   linked   to   virulence.   However,   the   deletions   did   not   affect   the   hypersensitive   response   or   in   planta   growth   of   the   resulting   strains.  Interestingly,  many  new  HrpL  regulon  members  appear  to  be  unrelated   to  the  T3SS  (based  on  their  annotations),  and  orthologs  for  some  of  these  can  be   identified   in   non-­-pathogenic  bacteria.   The   connection  of  these  new  HrpL  regulon   members  to  virulence  is  not  obvious.   The   HrpL   regulon   is   activated   as   a   result   of   a   chain   of   events,   most   of   which   are   not   well   understood.   It   is   known   that   RpoN,   which   controls   the   transcription   of   hrpL   in   DC3000,   is   required   for   virulence   in   several   bacterial   species.   Motivated   by   the   hypothesis   that   genes   are   coordinately   regulated   in   order   to   serve   a   strategic   purpose   (e.g.,   virulence),   our   second   goal   was   to   look   for   other   genes   activated   by   RpoN   in   parallel   with   hrpL.   RpoN   ([sigma]54)   requires   specialized   enhancer-­-binding   proteins   (EBPs)   in   order   to   activate   transcription.   This   arrangement   presumably   allows   the   cell   to   respond   to   environmental   signals   by   modifying   the   transcription   of   particular   genes.   Using   ChIP-­-Seq   and   RNA-­-Seq,   we   identified   candidate   RpoN-­-dependent   genes   as   well   as   genes   that   were   differentially   expressed   under   hrp-­-inducing   conditions.   This   initial   survey   includes   more   than   200   likely   RpoN-­-regulated   genes   involved   in   flagella   biosynthesis,   energy   metabolism,   nitrogen   metabolism,   transport   and   binding   proteins,  and  small  noncoding  RNAs,  as  well  as  putative  regulatory  proteins  and   EBPs.  Among  the  genes  that  were  differentially  regulated  between   hrp-­-inducing   and   repressing   conditions,   more   than   one   dozen   appear   to   be   regulated   by   RpoN   and  are  therefore  potentially  important  in  functions  related  to  plant  association   or  virulence.

Rhodopseudomonas palustris is a metabolically versatile phototrophic α-proteobacterium. The organism experiences a wide range of stresses in its environment and during metabolism. The oxidative an pH stresses of four ECF (extracytoplasmic function) σ-factors are investigated. Three of these, σ0550, σ1813, and σ1819 show responses to light-generated singlet oxygen and respiration-generated superoxide reactive oxygen species (ROS). The EcfG homolog, σ4225, shows a high response to superoxide and acid stress. Two proteins, one containing the EcfG regulatory sequence, and an alternative exported catalase, KatE, are presented to be regulated by σ4225. Transcripts of both genes show similar responses to oxidative stress compared to σ4225, indicating it is the EcfG-like σ-factor homolog and controls the global stress response in R. palustris.

Brucella ovis is a sexually transmitted, facultatively anaerobic, intracellular bacterial pathogen of sheep (Ovis aries) and red deer (Cervus elaphus). Brucella spp. infect primarily by penetrating the mucosa and are phagocytized by host macrophages, where survival and replication occurs. At least in some species, it has been shown that entry into stationary phase is necessary for successful infection. Brucella, like other alphaproteobacteria, lack the canonical stationary phase sigma factor ?s. Research on diverse members of this large phylogenetic group indicate the widespread presence of a conserved four-gene set including an alternative ECF sigma factor, an anti-sigma factor, a response regulator (RR), and a histidine kinase (HK). The first description of the system was made in Methylobacterium extorquens where the RR, named PhyR, was found to regulate the sigma factor activity by sequestering the anti-sigma factor in a process termed "sigma factor mimicry." These systems have been associated with various types of extracellular stress responses in a number of environmental bacteria. I hypothesized that homologous genetic sequences (Bov_1604-1607), which are similarly found among all Brucella species, may regulate survival functions during pathogenesis. To further explore the involvement of this system to conditions analogous to those occurring during infection, pure cultures of B. ovis cells were subjected to environments of pH (5 and 7) for 15, 30, and 45 minutes and oxidative (50mM H2O2) stress, or Spermine NONOate for 60 minutes. RNA was extracted and converted to cDNA andchanges in transcript levels of the sigma factor Bov1605 were measured using qPCR. Preliminary results indicate that under the exposure to Spermine NONOate there was little change in expression, but under oxidative stress expression of the sigma factor Bov1605 was 4.68-fold higher than that expressed under normal conditions. These results suggest that the sigma factor Bov1605 may be involved in oxidative stress defense during infection. Under acid stress (pH5), Bov1605 was found to be upregulated at 15 and 30 minutes, but after 45 and 60 minutes the time decreased.

The transition from the initial colonization stage into the chronic phase of infection in cystic fibrosis is associated with the conversion of Pseudomonas aeruginosa from the nonmucoid to mucoid, exopolysaccharide alginate-overproducing form. The upregulation of alginate production in mucoid P. aeruginosa strains occurs via the activation of the alternative sigma factor AlgU due to mutations in mucA or mucB , genes encoding negative regulators of algU. In this study we report the molecular characterization of the newly identified genes algW, mucD, and mucC. In addition, we demonstrate that the conversion into the mucoid phenotype in P. aeruginosa can be caused via a second, AlgU-independent pathway. The predicted primary structure of AlgW and MucD is homologous to the Escherichia coli serine protease HtrA and are negative regulators of the mucoid phenotype. Inactivation of mucD or algW results in the production of alginate. However, the effect on mucoidy via loss of algW is subtle and requires growth in the presence of the superoxide generator paraquat. Inactivation of mucC in the wild-type nonmucoid P. aeruginosa strain PAO1 did not cause any overt effect on alginate synthesis. Interestingly, loss of mucC in the mucA or mucB mutant background resulted in the mucoid phenotype when grown under conditions of increased osmolarity and temperature. Analysis of mucA in a collection of 53 mucoid P. aeruginosa clinical isolates revealed that the overall frequency of mutations was 84% suggesting that mucA is the preferential site for conversion to mucoidy. In a small percentage of strains, the conversion to mucoidy occurs independent of the AlgU pathway in which alginate production and transcription of the critical algD promoter depend on another alternative sigma factor, RpoN (sigma54). These two pathways are not independent as sigma54 represses the mucoid phenotype in AlgU-dependent strains via binding of RpoN to the algD promoter and interfering with sigmaE-dependent transcription. This phenomenon of two sigma factors undergoing antagonism, in addition to the negative regulation of each sigma subunit, may represent a higher, albeit more complex, level of control in the bacterial response to a multitude of environmental signals.