Identification of a Meningococcall-Glutamate ABC Transporter Operon Essential for Growth in Low-Sodium Environments

Abstract

GdhR is a meningococcal transcriptional regulator that was previously shown to positively control the expression of gdhA, encoding the NADP-specific L-glutamate dehydrogenase (NADP-GDH), in response to the growth phase and/or to the carbon source. In this study we used reverse transcriptase-PCR-differential display (to identify additional GdhR-regulated genes. The results indicated that GdhR, in addition to NADP-GDH, controls the expression of a number of genes involved in glucose catabolism by the Entner-Doudoroff pathway and in L-glutamate import by an unknown ABC transport system. The genes encoding the putative periplasmic substrate-binding protein (NMB1963) and the permease (NMB1965) of the ABC transporter were genetically inactivated. Uptake experiments demonstrated an impairment of L-glutamate import in the NMB1965-defective mutant in the absence or in the presence of a low sodium ion concentration. In contrast, at a sodium ion concentration above 60 mM, the uptake defect disappeared, possibly because the activity of a sodium-driven secondary transporter became predominant. Indeed, the NMB1965-defective mutant was unable to grow at a low sodium ion concentration (<20 mM) in a chemically defined medium containing L-glutamate and four other amino acids that supported meningococcal growth, but it grew when the sodium ion concentration was raised to higher values (>60 mM). The same growth phenotype was observed in the NMB1963-defective mutant. Cell invasion and intracellular persistence assays and expression data during cell invasion provided evidence that the L-glutamate ABC transporter, tentatively named GltT, was critical for meningococcal adaptation in the low-sodium intracellular environment.

Moraxella catarrhalis M35 Is a General Porin That Is Important for Growth under Nutrient-Limiting Conditions and in the Nasopharynges of Mice

“…Since it was not known whether the glycerol included by Monaco et al (31) for their study of Neisseria meningitidis would have the desired effect on M. catarrhalis, acetate and lactate were also added for some experiments. Lactate and acetate were chosen because M. catarrhalis has been shown to utilize both of these compounds as carbon sources (5,37).…”

“…An uptake assay following the method outlined by Monaco et al (31) with some modifications was used to assess the effect that deleting M35 has on glutamic acid uptake by M. catarrhalis. Isolates 4223 and 4223⌬M35 were grown overnight on CBA, harvested, and washed twice in buffer A (50 mM K 2 HPO 4 , 0.5 mM MgCl 2 [pH 6.9]), and by looking at the OD 400 , the concentration was adjusted to 1 ϫ 10 9 CFU/ml in buffer B (buffer A with 300 g/ml chloramphenicol).…”

TheNeisseria meningitidisCapsule Is Important for Intracellular Survival in Human Cells

“…The procedure for limited transcriptional analysis has been detailed previously (22,25). Briefly, a partial Sau3AI-restricted genomic library from serogroup B strain H44/76 was constructed.…”

“…Total RNAs were extracted from intracellular bacteria during the infection of HeLa cells or control bacteria grown in culture medium as described previously (22).…”

Effect of Host Lactate on Gonococci and Meningococci: New Concepts on the Role of Metabolites in Pathogenicity

“…The two roles of lactate might also operate for E. coli K-1 and group B streptococci, because like N. meningitidis, they also synthesize sialic acid (7,68). We are not aware of any other metabolites, such as glutamate (41), that have this dual role either on their own or in combination with another metabolite, but the observations of meningococci may be relevant to research on other organisms.…”