Competence regulation by oxygen availability and by Nox is not related to specific adjustment of central metabolism in Streptococcus pneumoniae

Abstract

In Streptococcus pneumoniae oxygen availability is a major determinant for competence development in exponentially growing cultures. NADH oxidase activity is required for optimal competence in cultures grown aerobically. The implication of oxidative metabolism and more specifically of Nox on central metabolism has been examined. Glycolytic flux throughout exponential growth revealed homolactic fermentation with a lactate production/glucose utilization ratio close to 2, whatever the aerobiosis level of the culture. Loss-of-function mutations in nox, which encodes NADH oxidase, did not change this trait. Consistently, mRNA levels of glyceraldehyde-3-phosphate dehydrogenase, L-lactate dehydrogenase, pyruvate oxidase, and NADH oxidase remained comparable to wild-type levels, as did the specific activities of key enzymes which control central metabolism. Competence regulation by oxygen involving the NADH oxidase activity is not due to significant modification of carbon flux through glycolysis. Failure to obtain loss-of-function mutation in L-ldh, which encodes the L-lactate dehydrogenase, indicates its essential role in pneumococci whatever their growth status.

FIG. 1

Kinetics of glucose utilization and lactate production during growth in CTM medium (1) under aerobiosis (A) and microaerobiosis (B) of the wild-type strain Cp1015, compared to that of the nox mutant strain Cp8056, grown under aerobiosis (C). Growth curves (D) were obtained by OD₄₀₀ measurements. Profiles of competence development throughout aerobic growth of Cp1015 and Cp8056 are also presented (E). For transformation tests, chromosomal DNA (1 μg/ml) carrying the rif₂₃ allele allowing resistance to rifampin (2 μg/ml) was used. Rif^r transformants were selected on plates, and their frequency in the population was calculated as described previously (1). For HPLC analysis, supernatants of cultures were filtered through 0.2-μm-pore-size Millipore filters.

FIG. 2

Effect of nox loss-of-function mutation and oxygen limitation on nox, L-ldh, spxB, and gap mRNA levels in cultures at an OD₄₀₀ of 0.1. Total-RNA preparations from competent cultures of the nox strain Cp8056 were subjected to Northern blot analysis with probes specific for ldh, nox, gap, and spxB. 16S rRNA was taken as a qualitative and quantitative internal control (see Table 1). A parallel analysis was performed on noncompetent cultures of the wild-type strain Cp1015 grown aerobically or microaerobically to an OD₄₀₀ of 0.1. Signals were quantified by densitometry, and ratios of specific mRNA to 16S rRNA are given. The experiments were repeated with independent cultures to test reproducibility.

FIG. 3

Evolution of gap mRNA levels throughout growth in cultures from the wild-type strain Cp1015 and the Nox₀ mutant Cp8056. Aliquots of cultures of Cp1015 (A) and Cp8056 (nox) (B) growing under aerobiosis in CTM medium (1) were withdrawn at 30-min intervals. Total-RNA preparations from these cultures were subjected to Northern blotting with a gap-specific probe. 16S rRNA was used as a qualitative and quantitative internal control. Signals were quantified by densitometry, and the 16S rRNA/gap mRNA ratio was calculated. For each strain, results were expressed relative to data obtained for cultures grown for 1 h 30 min, taken as 1. The experiments were repeated with independent cultures to test reproducibility.

FIG. 4

L-ldh mutagenesis by plasmid insertion duplication. The wild-type strain Cp1015 was transformed with the nonreplicative plasmids pLDH1, containing an internal fragment of L-ldh (A) and pLDH2, containing a fragment corresponding to the part of L-ldh encoding the C terminus of the enzyme (B). (C) Recombinant colonies were selected for resistance to spectinomycin (spcR) and verified by PCR amplification of the chromosome with oligonucleotides LDH11 and FPAM (lane 1, Eurogentec SmartLadder molecular weight marker, lane 2, PCR product of the pLDH2 plasmid, taken as a control; lanes 3 to 8, PCR products of transformed colonies).