Subcellular Min Oscillations as a Single-Cell Reporter of the Action of Polycations,Protamine, and Gentamicin on Escherichia coli

Background

In Escherichia coli, MinD-GFP fusion proteins show rapid pole to pole oscillations. The objective was to investigate the effects of extracellular cations on the subcellular oscillation of cytoplasmic MinD within Escherichia coli.

Methodology/Principal Findings

We exposed bacteria to the extracellular cations Ca⁺⁺, Mg⁺⁺, the cationic antimicrobial peptide (CAP) protamine, and the cationic aminoglycoside gentamicin. We found rapid and substantial increases in the average MinD oscillation periods in the presence of any of these polyvalent cations. For Ca⁺⁺ and Mg⁺⁺ the increases in period were transient, even with a constant extracellular concentration, while increases in period for protamine or gentamicin were apparently irreversible. We also found striking interdependence in the action of the small cations with protamine or gentamicin, distorted oscillations under the action of intermediate levels of gentamicin and Ca⁺⁺, and reversible freezing of the Min oscillation at high cationic concentrations.

Conclusions/Significance

Intracellular Min oscillations provide a fast single-cell reporter of bacterial response to extracellular polycations, which can be explained by the penetration of polycations into cells.     

Bacillus subtilis Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth

Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg²⁺ or Ca²⁺ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg²⁺ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg²⁺ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restore ponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg²⁺ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.     

Requirement of a Relatively High Threshold Level of Mg2+ for Cell Growth of a Rhizoplane Bacterium, Sphingomonas yanoikuyae EC-S001

Mg²⁺ is one of the essential elements for bacterial cell growth. The presence of the magnesium cation (Mg²⁺) in various concentrations often affects cell growth restoration in plant-associating bacteria. This study attempted to determine whether Mg²⁺ levels in Sphingomonas yanoikuyae EC-S001 affected cell growth restoration in the host plant and what the threshold level is. S. yanoikuyae EC-S001, isolated from the rhizoplane of spinach seedlings grown from surface-sterilized seeds under aseptic conditions, displayed uniform dispersion and attachment throughout the rhizoplane and phylloplane of the host seedlings. S. yanoikuyae EC-S001 did not grow in potato-dextrose broth medium but grew well in an aqueous extract of spinach leaves. Chemical investigation of the growth factor in the spinach leaf extract led to identification of the active principle as the magnesium cation. A concentration of ca. 0.10 mM Mg²⁺ or more allowed S. yanoikuyae EC-S001 to grow in potato-dextrose broth medium. Some saprophytic and/or diazotrophic bacteria used in our experiment were found to have diverse threshold levels for their Mg²⁺ requirements. For example, Burkholderia cepacia EC-K014, originally isolated from the rhizoplane of a Melastoma sp., could grow even in Mg²⁺-free Hoagland's no. 2 medium with saccharose and glutamine (HSG medium) and requires a trace level of Mg²⁺ for its growth. In contrast, S. yanoikuyae EC-S001, together with Bacillus subtilis IFO12113, showed the most drastic restoring responses to subsequent addition of 0.98 mM Mg²⁺ to Mg²⁺-free HSG medium. Our studies concluded that Mg²⁺ is more than just the essential trace element needed for cell growth restoration in S. yanoikuyae EC-S001 and that certain nonculturable bacteria may require a higher concentration of Mg²⁺ or another specific essential element for their growth.     

Divalent metal-dependent catalysis and cleavage specificity of CSP41, a chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase superfamily

CSP41 is a ubiquitous chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase (SDR) superfamily. To help elucidate the role of CSP41 in chloroplast gene regulation, the mechanisms that determine its substrate recognition and catalytic activity were investigated. A divalent metal is required for catalysis, most probably to provide a nucleophile for cleavage 5′ to the phosphodiester bond, and may also participate in cleavage site selection. This requirement distinguishes CSP41 from other Rossman fold-containing proteins from the SDR superfamily, including several RNA-binding proteins and endonucleases. CSP41 is active only in the presence of MgCl₂ and CaCl₂. Although Mg²⁺- and Ca²⁺-activated CSP41 cleave at identical sites in the single-stranded regions of a stem–loop-containing substrate, Mg²⁺-activated CSP41 was also able to cleave within the double-stranded region of the stem–loop. Mixed metal experiments with Mg²⁺ and Ca²⁺ suggest that CSP41 contains a single divalent metal-binding site which is non-selective, since Mn²⁺, Co²⁺ and Zn²⁺ compete with Mg²⁺ for binding, although there is no activity in their presence. Using site-directed mutagenesis, we identified three residues, Asn71, Asp89 and Asp103, which may form the divalent metal-binding pocket. The activation constant for Mg²⁺ (K_A,Mg = 2.1 ± 0.4 mM) is of the same order of magnitude as the stromal Mg²⁺ concentrations, which fluctuate between 0.5 and 10 mM as a function of light and of leaf development. These changes in stromal Mg²⁺ concentration may regulate CSP41 activity, and thus cpRNA stability, during plant development.     

The Adhesion-Associated sca Operon in Streptococcus gordonii Encodes an Inducible High-Affinity ABC Transporter for Mn2+ Uptake

ScaA lipoprotein in Streptococcus gordonii is a member of the LraI family of homologous polypeptides found among streptococci, pneumococci, and enterococci. It is the product of the third gene within the scaCBA operon encoding the components of an ATP-binding cassette (ABC) transporter system. Inactivation of scaC (ATP-binding protein) or scaA (substrate-binding protein) genes resulted in both impaired growth of cells and >70% inhibition of ⁵⁴Mn²⁺ uptake in media containing <0.5 μM Mn²⁺. In wild-type and scaC mutant cells, production of ScaA was induced at low concentrations of extracellular Mn²⁺ (<0.5 μM) and by the addition of ≥20 μM Zn²⁺. Sca permease-mediated uptake of ⁵⁴Mn²⁺ was inhibited by Zn²⁺ but not by Ca²⁺, Mg²⁺, Fe²⁺, or Cu²⁺. Reduced uptake of ⁵⁴Mn²⁺ by sca mutants and by wild-type cells in the presence of Zn²⁺ was abrogated by the uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting that Mn²⁺ uptake under these conditions was proton motive force dependent. The frequency of DNA-mediated transformation was reduced >20-fold in sca mutants. The addition of 0.1 mM Mn²⁺ to the transformation medium restored only partly the transformability of mutant cells, implying an alternate role for Sca proteins in the transformation process. Cells of sca mutants were unaffected in other binding properties tested and were unaffected in sensitivity to oxidants. The results show that Sca permease is a high-affinity mechanism for the acquisition of Mn²⁺ and is essential for growth of streptococci under Mn²⁺-limiting conditions.     

Characterization of Cadmium Uptake in Lactobacillus plantarum and Isolation of Cadmium and Manganese Uptake Mutants

Two different Cd²⁺ uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn²⁺ uptake system which also takes up Cd²⁺ and is induced by Mn²⁺ starvation. The calculated K_m and V_max are 0.26 μM and 3.6 μmol g of dry cell⁻¹ min⁻¹, respectively. Unlike Mn²⁺ uptake, which is facilitated by citrate and related tricarboxylic acids, Cd²⁺ uptake is weakly inhibited by citrate. Cd²⁺ and Mn²⁺ are competitive inhibitors of each other, and the affinity of the system for Cd²⁺ is higher than that for Mn²⁺. The other Cd²⁺ uptake system is expressed in Mn²⁺-sufficient cells, and no K_m can be calculated for it because uptake is nonsaturable. Mn²⁺ does not compete for transport through this system, nor does any other tested cation, i.e., Zn²⁺, Cu²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺, or Ni²⁺. Both systems require energy, since uncouplers completely inhibit their activities. Two Mn²⁺-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn²⁺ for growth as the parental strain. Mn²⁺ starvation-induced Cd²⁺ uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn²⁺ or Cd²⁺ accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn²⁺ and Cd²⁺ uptake system.     

Mechanisms of Cation Exchange by Pseudomonas aeruginosa PAO1 and PAO1 wbpL, a Strain with a Truncated Lipopolysaccharide

The ability of bacterial cells to sequester cations is well recognized, despite the fact that the specific binding sites and mechanistic details of the process are not well understood. To address these questions, the cation-exchange behavior of Pseudomonas aeruginosa PAO1 cells with a truncated lipopolysaccharide (LPS) (PAO1 wbpL) and cells further modified by growth in a magnesium-deficient medium (PAO1 wbpL − Mg²⁺) were compared with that of wild-type P. aeruginosa PAO1 cells. P. aeruginosa PAO1 cells had a negative surface charge (zeta potential) between pH 11 and 2.2, due to carboxylate groups present in the B-band LPS. The net charge on PAO1 wbpL cells was increasingly positive below pH 3.5, due to the influence of NH₃⁺ groups in the core LPS. The zeta potentials of these cells were also measured in Na⁺, Ca²⁺, and La³⁺ electrolytes. Cells in the La³⁺ electrolyte had a positive zeta potential at all pH values tested. Growing P. aeruginosa PAO1 wbpL in magnesium-deficient medium (PAO1 wbpL − Mg²⁺) resulted in an increase in its zeta potential in the pH range from 3.0 to 6.5. In cation-exchange experiments carried out at neutral pH with either P. aeruginosa PAO1 or PAO1 wbpL, the concentration of bound Ca²⁺ was found to decrease as the pH was reduced from 7.0 to 3.5. At pH 3.5, the bound Mg²⁺ concentration decreased sharply, revealing the activity of surface sites for cation exchange and their pH dependence. Infrared spectroscopy of attached biofilms suggested that carboxylate and phosphomonoester functional groups within the core LPS are involved in cation exchange.     

Prevalence of Streptococcus suis Genotypes in Wild Boars of Northwestern Germany

Invasive serotype 2 (cps2⁺) strains of Streptococcus suis cause meningitis in pigs and humans. Four case reports of S. suis meningitis in hunters suggest transmission of S. suis through the butchering of wild boars. Therefore, the objective of this study was to investigate the prevalence of potentially human-pathogenic S. suis strains in wild boars. S. suis was isolated from 92% of all tested tonsils (n = 200) from wild boars. A total of 244 S. suis isolates were genotyped using PCR assays for the detection of serotype-specific genes, the hemolysin gene sly, and the virulence-associated genes mrp and epf. The prevalence of the cps2⁺ genotype among strains from wild boars was comparable to that of control strains from domestic pig carriers. Ninety-five percent of the cps2⁺ wild boar strains were positive for mrp, sly, and epf*, the large variant of epf. Interestingly, epf* was significantly more frequently detected in cps2⁺ strains from wild boars than in those from domestic pigs; epf* is also typically found in European S. suis isolates from humans, including a meningitis isolate from a German hunter. These results suggest that at least 10% of wild boars in Northwestern Germany carry S. suis strains that are potentially virulent in humans. Additional amplified fragment length polymorphism analysis supported this hypothesis, since homogeneous clustering of the epf* mrp⁺ sly⁺ cps2⁺ strains from wild boars with invasive human and porcine strains was observed.     

Development, Characterization, and Application of a Cadmium-Selective Microelectrode for the Measurement of Cadmium Fluxes in Roots of Thlaspi Species and Wheat

A Cd²⁺-selective vibrating microelectrode was constructed using a neutral carrier-based Cd ionophore to investigate ion-transport processes along the roots of wheat (Triticum aestivum L.) and two species of Thlaspi, one a Zn/Cd hyperaccumulator and the other a related nonaccumulator. In simple Cd(NO₃)₂ solutions, the electrode exhibited a Nernstian response in solutions with Cd²⁺ activities as low as 50 nm. Addition of Ca²⁺ to the calibration solutions did not influence the slope of the calibration curve but reduced the detection limit to a solution activity of 1 μm Cd²⁺. Addition of high concentrations of K⁺ and Mg²⁺ to the calibration solution to mimic the ionic composition of the cytoplasm affected neither the slope nor the sensitivity of the electrode, demonstrating the pH-insensitive electrode's potential for intracellular investigations. The electrode was assayed for selectivity and was shown to be at least 1000 times more selective for Cd²⁺ than for any of those potentially interfering ions tested. Flux measurements along the roots of the two Thlaspi species showed no differences in the pattern or the magnitude of Cd²⁺ uptake within the time frame considered. The Cd²⁺-selective microelectrode will permit detailed investigations of heavy-metal ion transport in plant roots, especially in the area of phytoremediation.     

Quantitative studies of Mn(2+)-promoted specific and non-specific cleavages of a large RNA: Mn(2+)-GAAA ribozymes and the evolution of small ribozymes

Manganese (Mn²⁺) promotes specific cleavage at two major (I and III) and four minor (II, IV, V and VI) sites, in addition to slow non-specific cleavage, in a 659-nucleotide RNA containing the Cr.LSU group I intron. The specific cleavages occurred between G and AAA sequences and thus can be considered Mn²⁺-GAAA ribozymes. We have estimated rates of specific and non-specific cleavages under different conditions. Comparisons of the rates of major-specific and background cleavages gave a maximal specificity of approximately 900 for GAAA cleavage. Both specific and non-specific cleavages showed hyperbolic kinetics and there was no evidence of cooperativity with Mn²⁺ concentration. Interestingly, at site III, Mg²⁺ alone promoted weak, but the same specific cleavage as Mn²⁺. When added with Mn²⁺, Mg²⁺ had a synergistic effect on cleavage at site III, but inhibited cleavage at the other sites. Mn²⁺ cleavage at site III also exhibited lower values of K (Mn²⁺ requirement), pH-dependency and activation energy than did cleavage at the other sites. In contrast, the pH-dependency and activation energy for cleavage at site I was similar to non-specific cleavage. These results increase our understanding of the Mn²⁺-GAAA ribozyme. The implications for evolution of small ribozymes are also discussed.     

Ferrous Iron-Dependent Volatilization of Mercury by the Plasma Membrane of Thiobacillus ferrooxidans

Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe²⁺ medium (pH 2.5) supplemented with 6 μM Hg²⁺. In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 μM Hg²⁺. When incubated for 3 h in a salt solution (pH 2.5) with 0.7 μM Hg²⁺, resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe²⁺ was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30°C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe²⁺-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 μM Hg²⁺ and 1 mM Fe²⁺, plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe²⁺-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe²⁺-dependent mercury volatilization activity of the plasma membrane.     

Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme

Available evidence suggests that Mg²⁺ ions are involved in reactions catalyzed by hammerhead ribozymes. However, the activity in the presence of exclusively monovalent ions led us to question whether divalent metal ions really function as catalysts when they are present. We investigated ribozyme activity in the presence of high levels of Mg²⁺ ions and the effects of Li⁺ ions in promoting ribozyme activity. We found that catalytic activity increased linearly with increasing concentrations of Mg²⁺ ions and did not reach a plateau value even at 1 M Mg²⁺ ions. Furthermore, this dependence on Mg²⁺ ions was observed in the presence of a high concentration of Li⁺ ions. These results indicate that the Mg²⁺ ion is a very effective cofactor but that the affinity of the ribozyme for a specific Mg²⁺ ion is very low. Moreover, cleavage by the ribozyme in the presence of both Li⁺ and Mg²⁺ ions was more effective than expected, suggesting the existence of a new reaction pathway—a cooperative pathway—in the presence of these multiple ions, and the possibility that a Mg²⁺ ion with weak affinity for the ribozyme is likely to function in structural support and/or act as a catalyst.     

YscO of Yersinia pestis Is a Mobile Core Component of the Yop Secretion System

The Yersinia pestis low-Ca²⁺ response stimulon is responsible for the temperature- and Ca²⁺-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD, yscC, yscD, yscG, and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37°C in the absence of Ca²⁺. In this study, we characterized yscO of the Yop secretion (ysc) operon that contains yscN through yscU by determining the localization of its gene product and the phenotype of an in-frame deletion. The yscO mutant grew and expressed the same levels of Yops as the parent at 37°C in the presence of Ca²⁺. In the absence of Ca²⁺, the mutant grew independently of Ca²⁺, expressed only basal levels of V antigen and Yops, and failed to secrete these. These defects could be partially complemented by providing yscO in trans in the yscO mutant. Overexpression of YopM and V antigen in the mutant failed to restore the export of either protein, showing that the mutation had a direct effect on secretion. These results indicated that the yscO gene product is required for high-level expression and secretion of V antigen and Yops. YscO was found by immunoblot analysis in the soluble and membrane fractions of bacteria growing at 37°C irrespective of the presence of Ca²⁺ and in the culture medium in the absence of Ca²⁺. YscO is the only mobile protein identified so far in the Yersinia species that is required for secretion of V antigen and Yops.     

Heavy Metal Resistance Patterns of Frankia Strains

The sensitivity of 12 Frankia strains to heavy metals was determined by a growth inhibition assay. In general, all of the strains were sensitive to low concentrations (<0.5 mM) of Ag¹⁺, AsO₂¹⁻, Cd²⁺, SbO₂¹⁻, and Ni²⁺, but most of the strains were less sensitive to Pb²⁺ (6 to 8 mM), CrO₄²⁻ (1.0 to 1.75 mM), AsO₄³⁻ (>50 mM), and SeO₂²⁻ (1.5 to 3.5 mM). While most strains were sensitive to 0.1 mM Cu²⁺, four strains were resistant to elevated levels of Cu²⁺ (2 to 5 mM and concentrations as high as 20 mM). The mechanism of SeO₂²⁻ resistance seems to involve reduction of the selenite oxyanion to insoluble elemental selenium, whereas Pb²⁺ resistance and Cu²⁺ resistance may involve sequestration or binding mechanisms. Indications of the resistance mechanisms for the other heavy metals were not as clear.     

Influence of divalent cations on the conformation of phosphorothioate oligodeoxynucleotides: a circular dichroism study

Phosphorothioate oligodeoxynucleotides (ODNs) have been extensively investigated in vivo and in vitro for antisense control of gene expression. It has been shown that cellular uptake of phosphorothioate ODNs in some in vitro cell systems increases in the presence of divalent cations. In this work, we analyze the conformation of phosphorothioate ODNs and specific changes induced in it by various divalent cations using circular dichroism (CD) spectroscopy. CD data were obtained with several phosphorothioate ODNs in the absence and presence of the divalent cations Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺ and Mn²⁺. All CD spectra indicated stable conformations of the ODNs in solution. The spectra were strongly dependent on ODN sequence and composition. Some ODNs such as T₂₃ and another with ‘random’ distribution of bases showed CD spectra characteristic of B-form DNA. Other ODNs which had at least three consecutive guanines in their sequences exhibited spectra characteristic of parallel G-tetraplexes. CD spectra of antisense ODNs exhibited specific responses to divalent cations. Changes in the conformation were not simply due to ionic strength effects. Mn²⁺ diminished secondary structure in some ODNs. Group II divalent ions stabilized the parallel G-tetraplexes, and Mg²⁺ generally had the weakest stabilizing efficiency. Each sequence/ion combination had a specific response so these effects cannot be generalized. These sequence-dependent, divalent ion-sensitive, and structurally unique solution conformations may be related to ion-mediated ODN uptake.     

A Protein Phosphorylation Threshold for Functional Stacking of Plant Photosynthetic Membranes

Phosphorylation of photosystem II (PSII) proteins affects macroscopic structure of thylakoid photosynthetic membranes in chloroplasts of the model plant Arabidopsis. In this study, light-scattering spectroscopy revealed that stacking of thylakoids isolated from wild type Arabidopsis and the mutant lacking STN7 protein kinase was highly influenced by cation (Mg⁺⁺) concentrations. The stacking of thylakoids from the stn8 and stn7stn8 mutants, deficient in STN8 kinase and consequently in light-dependent phosphorylation of PSII, was increased even in the absence of Mg⁺⁺. Additional PSII protein phosphorylation in wild type plants exposed to high light enhanced Mg⁺⁺-dependence of thylakoid stacking. Protein phosphorylation in the plant leaves was analyzed during day, night and prolonged darkness using three independent techniques: immunoblotting with anti-phosphothreonine antibodies; Diamond ProQ phosphoprotein staining; and quantitative mass spectrometry of peptides released from the thylakoid membranes by trypsin. All assays revealed dark/night-induced increase in phosphorylation of the 43 kDa chlorophyll-binding protein CP43, which compensated for decrease in phosphorylation of the other PSII proteins in wild type and stn7, but not in the stn8 and stn7stn8 mutants. Quantitative mass spectrometry determined that every PSII in wild type and stn7 contained on average 2.5±0.1 or 1.4±0.1 phosphoryl groups during day or night, correspondingly, while less than every second PSII had a phosphoryl group in stn8 and stn7stn8. It is postulated that functional cation-dependent stacking of plant thylakoid membranes requires at least one phosphoryl group per PSII, and increased phosphorylation of PSII in plants exposed to high light enhances stacking dynamics of the photosynthetic membranes.     

Functional Similarity between Archaeal and Bacterial CorA Magnesium Transporters

The constitutively expressed CorA Mg²⁺ transporter is the major Mg²⁺ influx system of Salmonella typhimurium and Escherichia coli. Genomic sequence data indicated the presence of a homolog in the archaeal organism Methanococcus jannaschii. The putative M. jannaschii CorA was expressed in an Mg²⁺-transport-deficient strain of S. typhimurium to determine its functional characteristics. The archaeal CorA homolog is a functional Mg²⁺ uptake system when expressed in S. typhimurium and has properties which are highly similar to those of the normal CorA transporter of S. typhimurium despite having a low level of sequence identity with the protein and being expressed in a lipid membrane of quite different composition than normal. This implies that the overall function of the proteins is the same and further suggests that their structures are very similar.     

Insertional Inactivation of Determinants for Mg2+ and Co2+ Transport as a Tool for Screening Recombinant Lactococcus Species Clones

Insertional inactivation of the plasmid-encoded determinants for Mg²⁺ and Co²⁺ transport, orf18/corA, provides a tool for screening recombinant clones in Lactococcus, based on the observation that overexpression of orf18/corA results in cell growth inhibition on certain concentrations of CoCl₂. The lacticin 3147 immunity gene, ltnI, was used to insertionally inactivate orf18/corA. The resulting clones were capable of growth on concentrations of CoCl₂ that were inhibitory to the parent strain. Since only 3 of 17 lactococcal starters naturally harbor corA, the system has potential as a screen for selecting recombinant lactococcal clones.