Dam controls O-Ag chain length in Salmonella enterica by regulating the expression of Wzz protein

SARNACKI, S.H.; C.L. MAROLDA; M. NOTO LLANA; M.N. GIACOMODONATO; M.A. VALVANO; M.C. CERQUETTI

Tucumán, Argentina

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB)

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-GB;} @page Section1 {size:595.3pt 841.9pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> DNA adenine methyltransferase (Dam) enzyme plays an important role in physiological processes such as DNA replication, mismatch repair and transposition. We reported earlier that a Salmonella enterica serovar Enteritidis dam mutant expressing a truncated Dam fails to agglutinate in the presence of specific antibodies against O9 polysaccharide. Here we investigate the participation of Dam in the LPS synthesis of Salmonella. LPS O antigen profiles of a dam null mutant (SEDdam) and the parental strain of S. Enteritidis were examined by electrophoresis and silver staining. Compared to the parental strain, SEDdamproduced LPS with shorter O antigen polysaccharide chains. Since Wzz is responsible for the chain length distribution of the O antigen we investigated whether Dam methylation is involved in regulating wzz expression. Western blot analysis showed that the relative amount of Wzz produced by SEDdamis three-fold lower than that of the parental strain. β-galactosidase activity assay performed in the dam mutant containing a chromosomal lacZY fusion to the wzz locus, showed a reduction of 50 % in logarithmic phase and 25 % in stationary phase compared to the parental strain. These results were further confirmed by RT-PCR showing that wzz gene transcription was three-fold lower in the dam mutant compared with the parental strain. Next, we investigated whether increased expression of wzz in SEDdam could restore the wild type O antigen banding pattern. Parental and SEDdam strains were transformed with pwzz plasmid bearing the wzz gene under the regulation of tetracycline resistance gene promoter. The LPS pattern of SEΔdam/pwzz was similar to that found in the parental strain. It is known that RcsB and PmrA proteins regulate the expression of wzz gene; interestingly, we found that the rcsB mutant of S. Enteritidis shows an LPS pattern similar to that of the dam mutant. For that reason we investigated whether LPS pattern is affected in SEDdam mutant with an RcsB and PmrA overproducing background. To this purpose, rcsB and pmrA genes were cloned into pUC18 and electroporated in SEDdam mutant and wild type strain. We observed that the O antigen pattern in SEDdam RcsB overproducer was similar to that shown by the parental strain. On the contrary, the overproduction of PmrA did not modify the LPS chain length in SEDdam mutant. Interestingly, the expression of both pmrA and rcsB genes measured by RT-PCR was reduced in the dam mutant background. Our results demonstrate that wzz gene expression is downregulated in S. Enteritidis dam mutant, indicating that Dam methylation activates its expression. Moreover, Dam methylation would modulate  LPS O antigen synthesis through the regulation of the RcsC/YojN/RcsB two-component system.