Carbonic Anhydrase Is Essential for Streptococcus pneumoniae Growth in Environmental Ambient Air

The respiratory tract pathogen Streptococcus pneumoniae needs to adapt to the different levels of carbon dioxide (CO₂) it encounters during transmission, colonization, and infection. Since CO₂ is important for various cellular processes, factors that allow optimal CO₂ sequestering are likely to be important for pneumococcal growth and survival. In this study, we showed that the putative pneumococcal carbonic anhydrase (PCA) is essential for in vitro growth of S. pneumoniae under the CO₂-poor conditions found in environmental ambient air. Enzymatic analysis showed that PCA catalyzes the reversible hydration of CO₂ to bicarbonate (HCO₃⁻), an essential step to prevent the cellular release of CO₂. The addition of unsaturated fatty acids (UFAs) reversed the CO₂-dependent in vitro growth inhibition of S. pneumoniae strains lacking the pca gene (Δpca), indicating that PCA-mediated CO₂ fixation is at least associated with HCO₃⁻-dependent de novo biosynthesis of UFAs. Besides being necessary for growth in environmental ambient conditions, PCA-mediated CO₂ fixation pathways appear to be required for intracellular survival in host cells. This effect was especially pronounced during invasion of human brain microvascular endothelial cells (HBMEC) and uptake by murine J774 macrophage cells but not during interaction of S. pneumoniae with Detroit 562 pharyngeal epithelial cells. Finally, the highly conserved pca gene was found to be invariably present in both CO₂-independent and naturally circulating CO₂-dependent strains, suggesting a conserved essential role for PCA and PCA-mediated CO₂ fixation pathways for pneumococcal growth and survival.The Gram-positive bacterium Streptococcus pneumoniae, or pneumococcus, is a human respiratory tract pathogen that contributes significantly to global mortality and morbidity. In addition, it is an important asymptomatic colonizer of the human nasopharynx, with carriage rates around 10% in adults and over 40% in children (6). Pneumococcal colonization and infection are closely linked, but knowledge of the factors that contribute to transmission, carriage, disease, and transition from carriage to disease is still limited. Research on components that physically contribute to host-pathogen interactions, such as capsular polysaccharides, adhesins, and toxins, has provided valuable insights into the process of pneumococcal pathogenesis (20). In contrast, the influence of environmental factors on pneumococcal growth and survival remains fairly unexplored.S. pneumoniae needs to adapt to various aerobic and anaerobic conditions, reflecting the different niches it occupies during transmission, colonization, and invasive disease. During niche transition, oxygen (O₂) levels change considerably. Levels of O₂ are 21% in ambient air, decrease to 10 to 15% in the alveoli of the lungs, and are about 5% in resting cells. In O₂-rich conditions, S. pneumoniae expresses pyruvate oxidase (SpxB), which generates acetyl-phosphate as a source of ATP and hydrogen peroxide (H₂O₂) for interspecies competition at the mucosal surfaces of the nasopharynx (41). The presence of O₂ is also a prerequisite for the pneumococcal X state (4, 14), which is a physiological condition that allows for genetic transformation and an adequate response to environmental stress (32). Recently, it was shown that the fatty acid (FA) content of the pneumococcal cell membrane (31) and the expression of 69 genes (8) change in response to the availability of O₂. Finally, changes in O₂ levels can also affect production of the polysaccharide capsule (48), which is the major pneumococcal virulence determinant.Similar to those of O₂, the levels of carbon dioxide (CO₂) vary considerably among the different pneumococcal niches inside and outside the host. Ambient levels of CO₂ in the environment are 0.038%, while CO₂ levels inside the human body, in particular in the lower respiratory tract, can reach 5% or more. The importance of this gaseous compound for S. pneumoniae is illustrated by the observation that the depletion of CO₂ from ambient air completely inhibits pneumococcal growth (21). Moreover, about 8% of all clinical isolates require a CO₂-enriched environment for growth in laboratory conditions (3). This intrinsic CO₂ dependence of S. pneumoniae and many other (micro)organisms is most likely related to an anabolic need for CO₂ or bicarbonate (HCO_3⁻) during biosynthesis of nucleic acids, amino acids, and FAs (1). Pathogens can often sequester CO₂ directly from host tissues, but in the absence of sufficient levels of extracellular CO₂, endogenous CO₂ needs to be enzymatically fixated. Carbonic anhydrases (CAs; EC 4.2.1.1) are enzymes that catalyze the reversible reaction CO₂ + H₂O ↔ HCO₃⁻ + H⁺. Because HCO₃⁻ cannot passively diffuse across biological membranes, its formation significantly delays the release of intracellular CO₂. At least five different classes of CAs have been described, and most eukaryotic, prokaryotic, and archaeal species express at least one CA class (39, 40).Genome analysis (39) has revealed that S. pneumoniae has one putative CA, a β-class CA that is highly conserved in all available pneumococcal genome sequences. Pneumococcal CA (PCA) is highly homologous to CAs in other streptococcal species, such as Streptococcus pyogenes. The closest nonstreptococcal PCA homologs are found in Mycobacterium species, while PCA homologs in other respiratory tract pathogens such as Neisseria meningitidis and Haemophilus influenzae are more divergent (40). The aim of this study was to investigate the functional characteristics of the pca gene and the encoded PCA enzyme in S. pneumoniae and to establish the relevance of PCA for pneumococcal growth and survival under CO₂-poor conditions in vitro. Further, we examined the importance of PCA during host-pathogen interaction.     

Why does Kluyveromyces lactis not grow under anaerobic conditions? Comparison of essential anaerobic genes of Saccharomyces cerevisiae with the Kluyveromyces lactis genome

Although some yeast species, e.g. Saccharomyces cerevisiae, can grow under anaerobic conditions, Kluyveromyces lactis cannot. In a systematic study, we have determined which S. cerevisiae genes are required for growth without oxygen. This has been done by using the yeast deletion library. Both aerobically essential and nonessential genes have been tested for their necessity for anaerobic growth. Upon comparison of the K. lactis genome with the genes found to be anaerobically important in S. cerevisiae, which yielded 20 genes that are missing in K. lactis, we hypothesize that lack of import of sterols might be one of the more important reasons that K. lactis cannot grow in the absence of oxygen.     

T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis

The soil bacterium Agrobacterium tumefaciens can transfer a part of its tumour-inducing (Ti) plasmid, the T-DNA, to plant cells. The virulence (vir) genes, also located on the Ti plasmid, encode proteins involved in the transport of T-DNA into the plant cell. Once in the plant nucleus, T-DNA is able to integrate into the plant genome by an illegitimate recombination mechanism. The host range of A. tumefaciens is not restricted to plant species. A. tumefaciens is also able to transfer T-DNA to the yeast Saccharomyces cerevisiae. In this paper we demonstrate transfer of T-DNA from A. tumefaciens to the yeast Kluyveromyces lactis. Furthermore, we found that T-DNA serves as an ideal substrate for gene targeting in K. lactis. We have studied the efficiency of gene targeting at the K. lactis TRP1 locus using either direct DNA transfer (electroporation) or T-DNA transfer from Agrobacterium. We found that gene targeting using T-DNA was at least ten times more efficient than using linear double-stranded DNA introduced by electroporation. Therefore, the outcome of gene targeting experiments in some organisms may depend strongly upon the DNA substrate used. Received: 11 May 1998 / Accepted: 16 October 1998     

Association of connexin43 with a receptor protein tyrosine phosphatase

Connexin-43(Cx43)-based gap junctional communication is transiently inhibited by certain G protein-coupled receptor agonists, including lysophosphatidic acid, endothelin and thrombin. Our previous studies have implicated the c-Src protein tyrosine kinase in mediating closure of Cx43 based gap junctions. Pervanadate, an inhibitor of protein tyrosine phosphatases, mimics activated Src in inhibiting Cx43 gap junctional communication, apparently by promoting tyrosine phosphorylation of the Cx43 C-terminal tail. However, the identity of the protein tyrosine phosphatase(s) that may normally prevent Src-induced gap junction closure is unknown. Receptor-like protein tyrosine phosphatases that mediate homotypic cell-cell interaction are attractive candidates. Here we show that receptor protein tyrosine phosphatase mu (RPTPmu) interacts with Cx43 in diverse cell systems. We find that the first catalytic domain of RPTPmu binds to Cx43. Our results support a model in which RPTPmu, or a closely related protein tyrosine phosphatase, interacts with the regulatory C-terminal tail of Cx43 to prevent Src-mediated closure of Cx43 gap junctional channels.     

Mutational analysis of centromeric DNA elements of Klayveromyces lactis and their role in determining the species specificity of the highly homologous centromeres from K. lactis and Saccharomyces cerevisiae

The centromere of Kluyveromyces lactis was delimited to a region of approximately 280 bp, encompassing KICDEI, II, and III. Removal of 6 bp from the right side of KlCDEIII plus flanking sequences abolished centromere function, and removal of 5 bp of KICDEI and flanking sequences resulted in strongly reduced centromere function. Deletions of 20–80 bp from KlCDEII resulted in a decrease in plasmid stability, indicating that KlCDEII must have a certain length for proper centromere function. Centromeres of K. lactis do not function in Saccharomyces cerevisiae and vice versa. Adapting the length of K1CDEII to that of ScCDEII did not improve KlCEN function in S. cerevisiae, while doubling the ScCDEII length did not improve ScCEN function in K. lactis. Thus the difference in CDEII length is not in itself responsible for the species specificity of the centromeres from each of the two species of budding yeast. A chimeric K. lactis centromere with ScCDEIII instead of KlCDEIII was no longer functional in K. lactis, but did improve plasmid stability in S. cerevisiae, although to a much lower level then a wild-type ScCEN. This indicates that the exact CDEIII sequence is important, and suggests that the flanking AT-rich CDEII has to conform to specific sequence requirements.     

Mutation scanning of the NF2 gene: an improved service based on meta-PCR/sequencing, dosage analysis, and loss of heterozygosity analysis

We describe the development and implementation of a neurofibromatosis type 2 (NF2) mutation scanning service based on novel techniques. All 17 exons of the NF2 gene are amplified in four polymerase chain reaction (PCR) reactions, using the meta-PCR technique to link the NF2 exons into chimeric concatamers. The meta-PCR products are then scanned for point mutations by direct sequencing. A four-exon dosage assay is used to test for large deletion/duplication mutations. In certain cases when tumour studies are necessary, these techniques are also combined with loss of heterozygosity analysis with three highly polymorphic microsatellite markers located within or close to the NF2 gene. Over a period of 2 years, we have applied these techniques in a service setting to the analysis of 271 patient samples (245 lymphocyte DNA; 26 schwannoma DNA). Meta-PCR and sequencing identified 90 point mutations in the 271 blood and tumor samples, 48 of which have not been reported previously. Dosage analysis identified large deletions in 12 of the lymphocyte DNA samples. In addition, over 84% of mutations were identified in 23 schwannoma DNA samples in which complete analysis was possible. Adoption of this novel strategy has increased the overall mutation detection rate in familial NF2 cases to 88% and sporadic NF2 cases to 59%. It has also allowed us to decrease our reporting turnaround times, and because of a low overall failure rate, permitted the running of an efficient and cost-effective service.     

The cytochrome c domain of dimeric cytochrome cd(1) of Paracoccus pantotrophus can be produced at high levels as a monomeric holoprotein using an improved c-type cytochrome expression system in Escherichia coli

Cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is a dimer; within each monomer there is a largely alpha-helical domain that contains the c-type cytochrome centre. The structure of this domain changes significantly upon reduction of the heme iron, for which the ligands change from His17/His69 to Met106/His69. Overproduction, using an improved Escherichia coli expression system, of this c-type cytochrome domain as an independent monomer is reported here. The properties of the independent domain are compared with those when it is part of dimeric holo or semi-apo cytochrome cd(1).     

Red fluorescent protein (DsRed) as a reporter in Saccharomyces cerevisiae

We describe the utilization of a red fluorescent protein (DsRed) as an in vivo marker for Saccharomyces cerevisiae. Clones expressing red and/or green fluorescent proteins with both cytoplasmic and nuclear localization were obtained. A series of vectors are now available which can be used to create amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) fusions with the DsRed protein.