The mobilization of plasmids from gram-negative Escherichia coli to gram-positive Brevibacterium lactofermentum, mediated by P-type transfer functions, was used to construct disrupted mutants blocked specifically in the homoserine branch of the aspartate pathway. The mutant strain B. lactofermentum R31 showed an efficiency of conjugal transfer two to three orders of magnitude higher than that of the wild-type strain B. lactofermentum ATCC 13869. The hom- and thrB-disrupted mutants of B. lactofermentum ATCC 13869 were lysine overproducers. B. lactofermentum R31 mutants do not overproduce lysine because R31 is an alanine-overproducing strain and channels the pyruvate needed for lysine biosynthesis to the production of alanine.

The minimal region for autonomous replication of pBL1, a 4.5-kb cryptic plasmid of Brevibacterium lactofermentum ATCC 13869 that has been used to construct a variety of corynebacterium vectors, was shown to be contained on a 1.8-kb HindII-SphI DNA fragment. This region contains two open reading frames (ORFs) (ORF1 and ORF5) which are essential for pBL1 replication in B. lactofermentum. Accumulation of single-strand intermediates in some of the constructions indicates that plasmid pBL1 replicates via the rolling circle replication model; its plus strand and minus strand were identified by hybridization with two synthetic oligonucleotide probes complementary to each pBL1 strand. ORF1 seems to encode the Rep protein and showed partial homology with sequences for Rep proteins from Streptomyces plasmids which replicate via rolling circle replication such as pUJ101, pSB24, and pJVl.pBL1 is a multicopy plasmid isolated from Brevibacterium lactofermentum ATCC 13869 (19) and later described as pAM330 (14) Streptomyces lividans. pUL61 was unstable in B. lactofermentum, and two stable deletion derivatives (pUL330 and pUL340) appeared after B. lactofermentum had been transformed with pUL61. A similar instability has also been observed in most other small plasmids from gram-positive bacteria. The main characteristic of plasmids showing instability is their mode of replication via the rolling circle replication (RCR) mechanism. RCR plasmids were classified into four families according to homologies of their replication proteins (Rep) and the position of the double-stranded origin (DSO) (7). RCR plasmids have the information to synthesize their own Rep protein. The Rep protein initiates replication by nicking the DNA at the DSO sequence, after which replication of the plus strand begins. The newly generated single-stranded DNA (ssDNA) intermediates (plus strand) serve as a template for minus strand synthesis. The conversion of the ssDNA intermediates to duplex forms requires the recognition of another sequence, different and separated from the DSO sequence, called single-stranded origin (SSO). The absence or nonfunctionality of SSO results in the accumulation of ssDNA. The SSO sequence is rather specific, and it is usually recognized inefficiently in hosts different from the parental one (7, 15).We describe in this paper the identification and characterization of the region involved in replication functions of pBL1. Our results indicate that pBL1 belongs to the group of plasmids which replicate via the RCR mechanism. MATERIALS AND METHODSPlasmids, bacterial strains, and transformation conditions. Plasmids used in this study are listed in Table 1. B. lactofermentum BL31 (20), a pBL1-cured strain, and C. glutamicum ATCC 13032 were grown in Trypticase soy broth (TSB) medium and transformed by electroporation as described by Dunican and Shivnan (5). E. coli DH5ao was grown in L broth and transformed by the method of Hanahan (8).

Temperature and nutrient supply interactively control phytoplankton growth and productivity, yet the role of these drivers together still has not been determined experimentally over large spatial scales in the oligotrophic ocean. We conducted four microcosm experiments in the tropical and subtropical Atlantic (29°N-27°S) in which surface plankton assemblages were exposed to all combinations of three temperatures (in situ, 3 °C warming and 3 °C cooling) and two nutrient treatments (unamended and enrichment with nitrogen and phosphorus). We found that chlorophyll a concentration and the biomass of picophytoplankton consistently increase in response to nutrient addition, whereas changes in temperature have a smaller and more variable effect. Nutrient enrichment leads to increased picoeukaryote abundance, depressed Prochlorococcus abundance, and increased contribution of small nanophytoplankton to total biomass. Warming and nutrient addition synergistically stimulate light-harvesting capacity, and accordingly the largest biomass response is observed in the warmed, nutrient-enriched treatment at the warmest and least oligotrophic location (12.7°N). While moderate nutrient increases have a much larger impact than varying temperature upon the growth and community structure of tropical phytoplankton, ocean warming may increase their ability to exploit events of enhanced nutrient availability.

Corporate social responsibility (CSR) is a voluntary competitive strategy that is based upon social, economic, and environmental improvement in which the organisation is involved. Internationalisation, a type of corporate strategy, is a set of processes that help companies to expand globally to achieve the aim of improving their competitive position. Both of the strategies have become more important due to ever increasing globalisation, whose consequences modify economic and business environments, thus causing them to be more dynamic and competitive. This directly affects business management, thus companies increasingly consider the opinion of society, attempting to gain stakeholders’ trust through effective CSR management. In this context, this paper aims to analyse CSR and internationalisation strategies and their possible connection from a theoretical viewpoint. From a practical viewpoint, the relationship between both strategies is analysed while using a sample of Spanish listed companies.

Temperature and nutrient supply are key factors that control phytoplankton ecophysiology, but their role is commonly investigated in isolation. Their combined effect on resource allocation, photosynthetic strategy, and metabolism remains poorly understood. To characterize the photosynthetic strategy and resource allocation under different conditions, we analyzed the responses of a marine cyanobacterium (Synechococcus PCC 7002) to multiple combinations of temperature and nutrient supply. We measured the abundance of proteins involved in the dark (RuBisCO, rbcL) and light (Photosystem II, psbA) photosynthetic reactions, the content of chlorophyll a, carbon and nitrogen, and the rates of photosynthesis, respiration, and growth. We found that rbcL and psbA abundance increased with nutrient supply, whereas a temperature‐induced increase in psbA occurred only in nutrient‐replete treatments. Low temperature and abundant nutrients caused increased RuBisCO abundance, a pattern we observed also in natural phytoplankton assemblages across a wide latitudinal range. Photosynthesis and respiration increased with temperature only under nutrient‐sufficient conditions. These results suggest that nutrient supply exerts a stronger effect than temperature upon both photosynthetic protein abundance and metabolic rates in Synechococcus sp. and that the temperature effect on photosynthetic physiology and metabolism is nutrient dependent. The preferential resource allocation into the light instead of the dark reactions of photosynthesis as temperature rises is likely related to the different temperature dependence of dark‐reaction enzymatic rates versus photochemistry. These findings contribute to our understanding of the strategies for photosynthetic energy allocation in phytoplankton inhabiting contrasting environments.