Characterization of the dapA - nlpB Genetic Locus Involved in Regulation of Swarming Motility, Cell Envelope Architecture, Hemolysin Production, and Cell Attachment Ability in Serratia marcescens

Abstract

Swarming migration of Serratia marcescens requires both flagellar motility and cellular differentiation and is a population-density-dependent behavior. While the flhDC and quorum-sensing systems have been characterized as important factors regulating S. marcescens swarming, the underlying molecular mechanisms are currently far from being understood. Serratia swarming is thermoregulated and is characterized by continuous surface migration on rich swarming agar surfaces at 30°C but not at 37°C. To further elucidate the mechanisms, identification of specific and conserved regulators that govern the initiation of swarming is essential. We performed transposon mutagenesis to screen for S. marcescens strain CH-1 mutants that swarmed at 37°C. Analysis of a "precocious-swarming" mutant revealed that the defect in a conserved dapA Sm -nlpB Sm genetic locus which is closely related to the synthesis of bacterial cell wall peptidoglycan is responsible for the aberrant swarming phenotype. Further complementation and gene knockout studies showed that nlpB Sm , which encodes a membrane lipoprotein, NlpB Sm , but not dapA Sm , is specifically involved in swarming regulation. On the other hand, dapA Sm but not nlpB Sm is responsible for the determination of cell envelope architecture, regulation of hemolysin production, and cellular attachment capability. While the nlpB Sm mutant showed similar cytotoxicity to its parent strain, the dapA Sm mutant significantly increased in cytotoxicity. We present evidence that DapA Sm is involved in the determination of cell-envelope-associated phenotypes and that NlpB Sm is involved in the regulation of swarming motility.

Pirin Regulates Pyruvate Catabolism by Interacting with the Pyruvate Dehydrogenase E1 Subunit and Modulating Pyruvate Dehydrogenase Activity

“…After purification, GST protein pull-down assay was performed. Separation of captured proteins by SDS-PAGE highlighted three major bands with predicted molecular masses of 99 kDa (band a), 76 kDa (band b), and 50 kDa (band c) after comparison with the negative controls (GST-nlpB Sm [28] fusion and GST protein only) (Fig. 2).…”

“…The spent supernatants were then concentrated and analyzed by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to a polyvinylidene difluoride membrane (Amersham) and incubated in blocking buffer (5% milk, 0.1% Tween 20) for 1 h. Further incubations with anti-His monoclonal antibody or anti-pirin Sm polyclonal antibodies were then performed in blocking buffer for 1 h at room temperature, followed by treatment with horseradish peroxidase-conjugated anti-rabbit second antibody for another 1 h before development and X-ray film exposure (28).…”

“…For construction of the pirin Sm gene mutant, a protocol was designed for specific insertion of a 2-kb streptomycin (Sm)-resistant ⍀ cassette, excised from pHP45⍀, into the pirin Sm gene in S. marcescens CH-1 (23,28). Briefly, the 5Ј region of the pirin Sm gene was amplified by PCR using primer pair Pirk1 and Pirk2, TA cloned into pCR 2.1 (Invitrogen), and excised as a SalI/HindIII fragment.…”

“…Previous studies on selection for precocious-swarming mu-tants derived from Serratia marcescens CH-1 by transposon mutagenesis (15,28) identified a mutant strain in which a pirin gene homolog was inserted by use of a mini-Tn5 transposon (P.-C. Soo and H.-C. Lai, unpublished data). In comparison to human pirin, which is a 32-kDa protein consisting of 290 amino acids, and to E. coli pirin (YhhW), which is a 25.4-kDa protein with 231 amino acids, bioinformatic analyses identified a 312-amino-acid, 35-kDa pirin ortholog (pirin Sm ) in S. marcescens strain Db11 (Sanger Institute; http://www.sanger.ac.uk/cgi-bin /BLAST/submitblast/s_marcescens).…”

Regulation of Swarming Motility andflhDCSmExpression by RssAB Signaling inSerratia marcescens

“…In Serratia spp., swarming requires close interactions between the environment and the bacterial cells, as well as among the cells, in order to develop a high degree of complex cell coordination within the swarming colony (2,9,13,26,33,35). Previous studies on the regulation of swarming showed that bacterial flagellar, quorum-sensing, and two-component systems are important for swarming (3,7,33).…”