Regulation of LIC10368 expression by environmental factors. (A) RT-PCR analysis of LIC10368 transcripts upon culture-attenuation. RNA was extracted from low- and high-passage L. interrogans strains. Above, low-passage L. interrogans serovar Pomona strain LPF (passages 1 and 4, P1 and P4) and high-passage L. interrogans serovar Pomona strain Pomona (HP). Below, low-passage L. interrogans serovar Canicola strain LO4 (passages P1 and P3) and high-passage L. interrogans serovar Canicola strain Hond Utrechet IV (HP). (B) Regulation of LIC10368 transcript levels by osmolarity. Cultures of L. interrogans serovar Pomona strain LPF grown in EMJH with 1% rabbit serum were centrifuged and resuspended in fresh EMJH medium or in EMJH containing 120 mM NaCl. Cultures were incubated for 24 h before being harvested for RNA isolation. RNA was also obtained from a culture grown under our standard laboratory conditions (10% rabbit serum supplemented with amino acids and salts). LIC10368 transcripts obtained from cultures with 1% serum (1%), 1% serum + 120 mM NaCl (1% + NaCl) and 10% serum + amino acids and salts referred as enriched medium, EM (10% EM). 16S ribosomal cDNA fragments (1,042-bp) were co-migrated in all lanes. LIC12099 (lipL53 gene) transcripts were included as a positive control (1%, 1% + NaCl). (C) The effect of temperature on LIC10368 transcript levels was assessed by culturing leptospires at 20ºC, 29ºC and 37ºC. Additional cultures grown at 29ºC and at 37ºC were shifted overnight to 37ºC and to 39ºC, respectively, in order to simulate conditions encountered by bacteria upon entry into the host and in a febrile stage. Optical densities of LIC10368 and LIC12099 transcripts were normalized for each sample with corresponding 16S ribosomal densitometry data to obtain the relative expression levels. RT+: reverse transcriptase present in the reaction; RT-: reverse transcriptase omitted; M: molecular mass markers.